EPHA4-positive human adult pancreatic endocrine progenitor cells

ABSTRACT

The invention relates to the discovery of a selective cell surface marker that permits the selection of a unique subset of pancreatic stem cells having a high propensity to differentiate into insulin-producing cells or into insulin-producing cell aggregates.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/237,094, filed Sep. 27, 2005, which is hereby incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name 596572000510SEQLIST.txt,date recorded: Sep. 22, 2010, size: 1 KB).

BACKGROUND OF THE INVENTION

Curing type I diabetes would require either the regeneration or thereplacement of insulin-producing cells. Islet transplantation has beenextensively investigated as a treatment, but the shortage of human donorsources and the low efficiency of islet isolation has largely hamperedthis therapy. Alternative sources of islets would be desirable. Onemajor focus of study has been the ex vivo cultivation, expansion anddifferentiation of functional human endocrine cells for clinicalapplications.

Stem cells offer great potential for cell-replacement therapy. Mouseembryonic stem cells injected into rat striatum were shown to matureinto dopaminergic neurons leading to partial recovery in a rat model ofParkinson's disease. Haematopoietic stem cells that regenerate bloodcells after bone marrow transplantation are in wide clinical use such asin the treatment of leukemia. Recently, the prospects for using adultstem cells in medical treatments were heightened by Canadian cardiacsurgeons, who reported that injecting bone marrow cells into the heartcan boost its function.

Advances in defining the molecular basis of early pancreogenesis havecontributed to an understanding of the process of regeneration thatoccurs in animal models of pancreatic injury and diabetes. However,pancreatic progenitor cell populations remain poorly defined and thesubject of considerable debate. The identity of the islet progenitorcells has remained elusive. Identification of the markers that aid theisolation and purification of islet progenitor cell therefore isimportant to developing regenerated beta-cells in culture for subsequenttransplantation into diabetic patients.

Eph receptors, the largest subfamily of receptor tyrosine kinases(RTKs), are important mediators of cell-cell communication regulatingcell attachment, shape, and mobility. Eph signaling is crucial for thedevelopment of many developmental processes, including embryopatterning, angiogenesis and axon guidance. Emerging evidence alsosupports a role for these molecules in the formation of adult tissuesand organs, such as the nervous and cardiovascular systems.

Both Ephs and ephrins are membrane-bounded and their interaction atsites of cell-cell contact initiate unique bi-directional signalingcascades. Recent studies showed that signaling by Eph receptors controlsoocyte maturation in C. elegans by inhibition of MAPK activationdemonstrated that EphrinB1 forward and reverse signaling are requiredduring mouse development. Conditional deletion of EphrinB1 revealed thatEphrinB1 acts autonomously in neural crest cells and controls theirmigration. A mutation study in the PDZ binding domain indicated thatEphrinB1-induced reverse signaling is required in neural crestcell-derived tissue formation. Those results showed that EphrinB1 actsboth as a ligand and as a receptor in a tissue-specific manner duringembryognesis.

Combinatorial expression of Eph and Ephrins may define migration andpositioning in a wide spectrum of adult tissues. In the small intestine,β-catenin and TCF couple proliferation and differentiation to thesorting of cell populations through controlling the expression theEphB/EphrinB proteins. Eph proteins serve as cell surface markers formonitoring the cell proliferation and differentiation. Invasculogenesis, arteries and veins are morphologically, functionally andmolecularly very different. Notch-gridlock(grl) signaling pathway playimportant role in the development of arteries and veins. Inhibition ofgrl expression, by gene mutation or antisense RNA, ablates regions ofthe artery, and expands contiguous regions of the vein, proceed by anincrease in expression of the venous marker EphB4 receptor anddiminution of expression of the arterial marker EphrinB2.

The findings mentioned above show that Eph widely exists in differentepithelial tissues of different species. In an adult animal colon model,Eph expresses only in the proliferating and developing stages of theepithelial cells during normal tissue self renewal, not in stem cellsnor in mature cells. Eph also does not express in mesenchymal cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for obtaining cultures ofpropagating pancreatic cells and cultures of such cells.

In a first group of embodiments, the invention provides obtaining aculture of propagating pancreatic cells comprising isolating pancreaticcells from a pancreas; contacting the pancreatic cells with an EphA4binding reagent; selecting pancreatic cells that specifically bind tothe EphA4 binding reagent; and separating the selected pancreatic cellsfrom pancreatic cells that do not bind the EphA4 binding reagent toobtain a culture of propagating pancreatic cells. In some embodiments,the EphA4 binding reagent is labeled. In some embodiments, the step ofselecting is done by fluorescence activated cell sorting. In someembodiments, the step of selecting is done by panning. In someembodiments, the EphA4-binding reagent is an antibody that specificallybinds to the EphA4 protein. In some embodiments, the pancreas is from ahuman. In some embodiments, the method further comprises propagating thecells of step and differentiating the cells into an aggregate of insulinproducing cells. In some embodiments, the step of differentiating thecells comprises culturing the cells on plates coated with collagen IV.In some embodiments, the step of differentiating the cells comprisesculturing the cells in a media comprising a differentiation factor. Insome embodiments, the differentiation factor is selected from the groupconsisting of hepatocyte growth factor, keratinocyte growth factor, andexendin-4. In some preferred embodiments, the differentiation factor ishepatocyte growth factor. In some embodiments, the method furthercomprises contacting the pancreatic cells with a CD56 binding reagent;selecting pancreatic cells that specifically bind to the CD56 bindingreagent; and separating the selected pancreatic cells from pancreaticcells that do not bind the CD56 binding reagent to obtain a culture ofpropagating pancreatic cells. The sorting for cells that specificallybind to the CD56 binding reagent can be performed before contacting thepancreatic cells with the EphA4 binding reagent, or after cells bindingthe EphA4 binding reagent are separated from pancreatic cells that donot bind the EphA4 binding reagent.

In a further group of embodiments, the invention provides methods ofproducing an aggregate of insulin-producing pancreatic cells. Thesemethods comprise the steps of isolating pancreatic cells from apancreas; contacting the pancreatic cells with an EphA4 binding reagent;selecting pancreatic cells that specifically bind to the EphA4 bindingreagent; separating the selected pancreatic cells from pancreatic cellsthat do not bind the CD56 binding reagent to obtain a culture ofpropagating pancreatic cells; and differentiating the propagatingpancreatic cell culture into an aggregate of insulin producingpancreatic cells. In some embodiments, the EphA4 binding reagent islabeled. In some embodiments, the step of selecting is done byfluorescence activated cell sorting. In some embodiments, the step ofselecting is done by panning. In some embodiments, the EphA4-bindingreagent is an antibody that specifically binds to the EphA4 protein. Insome embodiments, the pancreas is from a human. In some embodiments, themethod further comprises propagating the cells of step anddifferentiating the cells into an aggregate of insulin producing cells.In some embodiments, the step of differentiating the cells comprisesculturing the cells on plates coated with collagen IV. In someembodiments, the step of differentiating the cells comprises culturingthe cells in a media comprising a differentiation factor. In someembodiments, the differentiation factor is selected from the groupconsisting of hepatocyte growth factor, keratinocyte growth factor, andexendin-4. In some preferred embodiments, the differentiation factor ishepatocyte growth factor. In some embodiments, the method furthercomprises contacting the pancreatic cells with a CD56 binding reagent;selecting pancreatic cells that specifically bind to the CD56 bindingreagent; and separating the selected pancreatic cells from pancreaticcells that do not bind the CD56 binding reagent to obtain a culture ofpropagating pancreatic cells. The sorting for cells that specificallybind to the CD56 binding reagent can be performed before contacting thepancreatic cells with the EphA4 binding reagent, or after cells bindingthe EphA4 binding reagent are separated from pancreatic cells that donot bind the EphA4 binding reagent.

In yet another group of embodiments, the invention provides methods ofproviding pancreatic endocrine function to a mammal in need of suchfunction, comprising the steps of isolating pancreatic cells from apancreas; contacting the pancreatic cells with an EphA4 binding reagent;selecting pancreatic cells that specifically bind to the EphA4 bindingreagent; separating the selected pancreatic cells from pancreatic cellsthat do not bind the EphA4 binding reagent to obtain a culture ofpropagating pancreatic cells; and implanting into the mammal thepropagating pancreatic cells in an amount sufficient to produce ameasurable amount of insulin in the mammal. In some embodiments, theEphA4 binding reagent is labeled. In some embodiments, the step ofselecting is done by fluorescence activated cell sorting. In someembodiments, the step of selecting is done by panning. In someembodiments, the EphA4-binding reagent is an antibody that specificallybinds to the EphA4 protein. In some embodiments, the pancreas is from ahuman. In some embodiments, the propagating pancreatic cellsdifferentiate into aggregates of insulin producing pancreatic cellsafter implantation into the mammal. In some embodiments, beforeimplantation into the mammal, the propagating pancreatic cell culture isdifferentiated into an aggregate of insulin producing pancreatic cells.In some embodiments, the step of differentiating the cells comprisesculturing the cells on plates coated with collagen IV. In someembodiments, the step of differentiating the cells comprises culturingthe cells in a media comprising a differentiation factor. In someembodiments, the differentiation factor is selected from the groupconsisting of hepatocyte growth factor, keratinocyte growth factor, andexendin-4. In some preferred embodiments, the differentiation factor ishepatocyte growth factor. In some embodiments, the mammal is a human. Insome embodiments, the method further comprises contacting the pancreaticcells with a CD56 binding reagent; selecting pancreatic cells thatspecifically bind to the CD56 binding reagent; and separating theselected pancreatic cells from pancreatic cells that do not bind theCD56 binding reagent to obtain a culture of propagating pancreaticcells. The sorting for cells that specifically bind to the CD56 bindingreagent can be performed before contacting the pancreatic cells with theEphA4 binding reagent, or after cells binding the EphA4 binding reagentare separated from pancreatic cells that do not bind the EphA4 bindingreagent.

In still another group of embodiments, the invention provides methods ofmonitoring a culture of propagating pancreatic cells by contacting thepancreatic cells with a EphA4 binding reagent; and determining thequantity of cells that exhibit EphA4 as a cell surface marker. In someembodiments, the detecting step is done by fluorescence activated cellsorting. In some embodiments, the EphA4 binding reagent is an antibodythat binds specifically to EphA4 protein. In some embodiments, thepancreas is from a human. In some embodiments, the methods furthercomprise the steps of contacting the pancreatic cells with a CD56binding reagent; selecting pancreatic cells that specifically bind tothe CD56 binding reagent; and determining the quantity of cells thatexhibit CD56 and EphA4 as cell surface markers.

In a further group of embodiments, the invention provides cell culturesproduced by the steps of: isolating pancreatic cells from a pancreas,contacting the pancreatic cells with an EphA4 binding reagent; selectingpancreatic cells that specifically bind to the EphA4 binding reagent;separating the selected pancreatic cells from pancreatic cells that donot bind the EphA4 binding reagent to obtain a culture of propagatingpancreatic cells. In some embodiments, the EphA4 binding reagent islabeled. In some embodiments, the step of selecting is done byfluorescence activated cell sorting. In some embodiments, the step ofselecting is done by panning. In some embodiments, the EphA4 bindingreagent is an antibody that specifically binds to the EphA4 protein. Insome embodiments, the pancreas is from a human. In some embodiments, thesteps of producing the cell culture further comprise contacting thepancreatic cells with a CD56 binding reagent; selecting pancreatic cellsthat specifically bind to the CD56 binding reagent; and separating theselected pancreatic cells from pancreatic cells that do not bind theCD56 binding reagent. In some embodiments, the steps of separating thecells for cells binding the CD56 binding reagent are performed beforecontacting the pancreatic cells with the EphA4 binding reagent and insome embodiments, they are performed after contacting the pancreaticcells with the EphA4 binding reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. This figure shows a flow chart for EphA4 sorting and cellculturing. In this and the other Figures, the letter “P” followed by anumber designates a given cell passage.

FIGS. 2 a-2 d. FIGS. 2 a-d show immunofluorescence (“IF”) and ICCstudies of expression of EphA4 in adult pancreas and primary pancreaticculture. FIG. 2 a. Adult human islet, paraffin section. IF EphA4staining, 100×. EphA4-positive cells were restricted to some isletcells; no positive staining is seen in acinar cells. FIG. 2 b. Adulthuman islet, paraffin section. bright field, 100×. FIG. 2 c. Adult humanislet, in culture. ABC EphA4 staining, 200×. Expression of EphA4 isfound in a subgroup of cultured pancreatic cells. FIG. 2 d. Adult humanislet, in culture. Phase, 100×.

FIG. 3. FIG. 3 shows a comparison of expression of insulin in EphA4+ andin EphA4− cells (in this Figure and in the others below that showexpression levels, the expression levels are shown as a ratio ofexpression of the expression of the gene under discussion compared tothe expression of beta-actin, a well known “housekeeping gene” oftenused in the art as a base for comparison of relative expression levels).Expression of insulin in EphA4-sorted cells was evaluated by real-timePCR. The mRNA levels of insulin in EphA4+ cells were 9-112 times higherthan those of EphA4− cells.

FIG. 4. FIG. 4 shows a comparison of expression of PDX-1 in EphA4+ andin EphA4− cells. Expression of PDX-1 in sorted cells was evaluated byreal-time PCR. EphA4+ cells showed 6-10 times higher mRNA levels ofPDX-1 than did EphA4− cells either just after sorting (P2) or during thefollowing passages (P3-P6). PDX-1 was expressed at a high level at earlypassage P2. The expression of PDX-1 gradually decreased as the number ofcell passages increased. After culturing in differentiation media, theexpression of PDX-1 returned to high levels in EphA4+ cells.

FIG. 5. FIG. 5 shows a comparison of expression of the pancreaticprogenitor marker Nkx2.2 (a homeodomain transcription factor expressedin early stages of pancreatic development) in EphA4+ and in EphA4−cells. Expression of Nkx2.2 in EphA4-sorted cells was evaluated byreal-time PCR. EphA4+ cells showed 2.5-16 times higher mRNA levels ofNkx2.2 than did EphA4− cells at different cell passages.

FIG. 6. FIG. 6 shows a comparison of expression of the pancreaticprogenitor marker Pax6 (a pancreatic progenitor marker that is a latefactor after neurogenin3 expression that is critical to development ofthe differentiated islet cell phenotype) in EphA4+ and in EphA4− cells.Expression of Pax6 in EphA4-sorted cells was evaluated by real-time PCR.EphA4+ cells showed 8.5-64 times higher mRNA levels of Pax6 than didEphA4− cells at different cell passages.

FIG. 7. FIG. 7 shows a flow chart for CD56 cell sorting and culturing.The presence of a cell marker on the sorted cells is indicated by apositive sign (“+”), its absence is indicated by a negative sign (“−”).

FIG. 8. FIG. 8 shows the expression of insulin in CD56-sorted cells.Expression of insulin in CD56-sorted cells was evaluated by real-timePCR. The mRNA levels of insulin in CD56+ cells were 4-12 times higherthan those of CD56− cells. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 9. FIG. 9 shows a comparison of expression of PDX-1 in CD56+ and inCD56− cells. Expression of PDX-1 in CD56-sorted cells was evaluated byreal-time PCR. CD56+ and CD56− cells showed equal expression of PDX-1 atP2, but the expression of PDX-1 gradually elevated as the number of cellpassages (P3-P5) increased and reached 26 times higher than CD56− cellsat P6. Presence of a cell marker on the sorted cells is indicated by apositive sign (“+”), its absence is indicated by a negative sign (“−”).

FIG. 10. FIG. 10 shows a comparison of expression of the pancreaticprogenitor marker Nkx2.2 in CD56+ and in CD56− cells. Expression ofNkx2.2 in CD56-sorted cells was evaluated by real-time PCR. CD56+ cellsshowed 1.6-7 times higher mRNA levels of Nkx2.2 than did CD56− cells atdifferent cell passages. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 11. FIG. 11 shows a comparison of expression of the pancreaticprogenitor marker Pax6 in CD56+ and in CD56− cells. Expression of Pax6in CD56-sorted cells was evaluated by real-time PCR. CD56+ cells showed4-7 times higher mRNA levels of Pax6 than did CD56− cells at differentcell passages. The presence of a cell marker on the sorted cells isindicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 12. FIG. 12 shows a flow chart of “double selecting” cells for thepresence of absence of both the EphA4 and CD56 markers. Presence of acell marker on the sorted cells is indicated by a positive sign (“+”),its absence is indicated by a negative sign (“−”).

FIGS. 13 a-d. FIGS. 13 a-d show the expression of insulin in EphA4 andCD56 double selected cells at different cell passages. FIG. 13 a: cellpassage 2 (“P2”). FIG. 13 b: cell passage 3. FIG. 13 c: cell passage 4.The horizontal axis shows both cell selections and different cultureconditions. FIG. 13 d: cell passage 6. The horizontal axis shows bothcell selections and different culture conditions. Expression of insulinwas evaluated by real-time PCR. As shown in FIGS. 13 c and d, the doublepositive cells maintained high expression of insulin through sequentialpassages with different culture conditions. The mRNA levels of insulinin CD56-EphA4 double positive cells were always around 2 to 5 timeshigher than either EphA4 or CD56 single positive selected cells duringthe passages. Presence of a cell marker on the sorted cells is indicatedby a positive sign (“+”), its absence is indicated by a negative sign(“−”).

FIGS. 14 a-d. FIGS. 14 a-d show a comparison of expression of thepancreatic gene PDX-1 in cells according to their expression of cellmarkers CD56 and EphA4 at various cell passages. FIG. 14 a:PDX-1/Beta-actin expression ratio at P2. FIG. 14 b: PDX-1/Beta-actinexpression ratio at P3. FIG. 14 c: PDX-1/Beta-actin expression ratio atP4. The horizontal axis shows both cell selections and different cultureconditions. FIG. 14 d: PDX-1/Beta-actin expression ratio at P6. Thehorizontal axis shows both cell selections and different cultureconditions. All Figures: expression of PDX-1 in cells was evaluated byreal-time PCR. EphA4/CD56 double positive cells showed higher expressionof PDX-1 throughout cell passages P2-P6. During cell passages P3-P6, theexpression of PDX-1 was higher in double positive cells than in EphA4single positive cells. At P2, double positive cells and EphA4 positivecells had similar levels of PDX-1 expression. The results show thatselection of double positive cells further enhances the pancreaticendocrine phenotype. Presence of a cell marker on the sorted cells isindicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIGS. 15 a-d. FIGS. 15 a-d show a comparison of expression of thepancreatic progenitor marker Nkx2.2 in cells according to theirexpression of cell markers CD56 and EphA4 at various cell passages. FIG.15 a: Nkx2.2/Beta-actin expression ratio at P2. FIG. 15 b:Nkx2.2/Beta-actin expression ratio at P3. FIG. 15 c: Nkx2.2/Beta-actinexpression ratio at P4. FIG. 15 d: Nkx2.2/Beta-actin expression ratio atP5. All Figures: expression of Nkx2.2 in cells was evaluated byreal-time PCR. EphA4/CD56 double positive cells showed higher expressionof Nkx2.2 throughout the cell passages. The results show that selectionof double positive cells further enriched for pancreatic progenitorcells over single selection. Presence of a cell marker on the sortedcells is indicated by a positive sign (“+”), its absence is indicated bya negative sign (“−”).

FIGS. 16 a-d. FIGS. 16 a-d show a comparison of expression of thepancreatic progenitor marker Pax6 in cells according to their expressionof cell markers CD56 and EphA4 at various cell passages. FIG. 16 a:Pax6/Beta-actin expression ratio at P2. FIG. 16 b: Pax6/Beta-actinexpression ratio at P3. FIG. 16 c: Pax6/Beta-actin expression ratio atP4. FIG. 16 d: Pax6/Beta-actin expression ratio at P5. All Figures:expression of Pax6 in cells was evaluated by real-time PCR. EphA4/CD56double positive cells showed higher expression of Pax6 throughout thecell passages. The results show that selection of double positive cellsfurther enriched for pancreatic progenitor cells over single selection.Presence of a cell marker on the sorted cells is indicated by a positivesign (“+”), its absence is indicated by a negative sign (“−”).

FIG. 17. FIG. 17 shows a flow chart for cell sorting, encapsulation, andtransplantation of cells with or without EphA4, CD56, or both cellmarkers, into diabetic C57 mice. Presence of a cell marker on the sortedcells is indicated by a positive sign (“+”), its absence is indicated bya negative sign (“−”).

FIG. 18. FIG. 18 shows the CD56 levels in sorted cells after variouscell passages. P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 19. FIG. 19 shows the EphA4 levels in sorted cells after variouscell passages. P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 20. FIG. 20 shows the insulin levels in sorted cells after variouscell passages. P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 21. FIG. 21 shows the PDX-1 levels in sorted cells after variouscell passages. P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 22. FIG. 22 shows GLUT2 levels in sorted cells after various cellpassages (GLUT2, or glucose transporter, type 2, is the major glucosetransporter isoform expressed in insulin-secreting pancreatic betacells). P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 23. FIG. 23 shows the glucagon levels in sorted cells after variouscell passages (glucagon is a linear peptide of 29 amino acids that has amajor role in maintaining normal levels of blood glucose). P0pre-sorting: cells in passage before sorting for cell markers. P0post-1st sorting, cells in passage just after sorting. P1 before secondsorting: first cell passage after 1st sorting, before second sorting.P1, Post-2nd sorting: first cell passage after sorting for both EphA4and CD56. Presence of a cell marker on the sorted cells is indicated bya positive sign (“+”), its absence is indicated by a negative sign(“−”).

FIG. 24. FIG. 24 shows the CK19 levels in sorted cells after variouscell passages. P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIG. 25. FIG. 25 shows the pancreatic amylase levels in sorted cellsafter various cell passages (pancreatic amylase is a pancreaticsaccharidase). P0 pre-sorting: cells in passage before sorting for cellmarkers. P0 post-1st sorting, cells in passage just after sorting. P1before second sorting: first cell passage after 1st sorting, beforesecond sorting. P1, Post-2nd sorting: first cell passage after sortingfor both EphA4 and CD56. Presence of a cell marker on the sorted cellsis indicated by a positive sign (“+”), its absence is indicated by anegative sign (“−”).

FIGS. 26 a and b. FIGS. 26 a and b show a comparison of expression ofinsulin in CD56/EphA4 single sorted cells and for cells sorted first forCD56 and then for EphA4 against cells sorted first for EphA4 and thenfor CD56. FIG. 26 a: Cells sorted first for CD56 at the end of P0 andthen with EphA4 at the end of P1. FIG. 26 b: Cells sorted first forEphA4 at the end of P0 and then for CD56 at the end of P1. As shown,insulin expression was determined by qRT-PCR at P0, P1 and P2. Presenceof a cell marker on the sorted cells is indicated by a positive sign(“+”), its absence is indicated by a negative sign (“−”).

FIGS. 27 a and b. FIGS. 27 a and b show a comparison of expression ofinsulin in CD56/EphA4 single sorted cells and for cells sorted first forCD56 and then for EphA4 against cells sorted first for EphA4 and thenfor CD56 after culturing in different media.

FIG. 27 a: Cells sorted first for CD56 at the end of P0 and then forEphA4 at the end of P1. FIG. 27 b: Cells sorted first for EphA4 at theend of P0 and then for CD56 at the end of P1. Both Figures: after thesecond sorting, the sorted cells were divided into two groups. One groupwas kept in medium SM95 (shown as “95” in the graph) and the other wastreated with differentiation medium (“DM,” in the graph) for three days.Insulin was tested for both groups at P2 by qRT-PCR. Insulin expressionwas twice as high in the group of cells treated with DM. Presence of acell marker on the sorted cells is indicated by a positive sign (“+”),its absence is indicated by a negative sign (“−”).

FIG. 28. FIG. 28 shows the changes in blood glucose of four diabeticmice which recovered from hyperglycemia after transplantation withencapsulated CD56 or EphA4 sorted human pancreatic cells, or withencapsulated EphA4-CD56 double selected human pancreatic cells, asdescribed in the Examples.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Surprisingly, it has now been discovered that the protein EphA4 is anextracellular marker for progenitors of pancreatic β cells. Sortingpancreatic cells for cells bearing the EphA4 extracellular markerresults in a population enriched in pancreatic progenitor cells.EphA4-positive pancreatic cells are capable of being propagated and canbe differentiated into aggregates of insulin producing pancreatic cells.Further, pancreatic cells bearing the EphA4 marker can bedouble-selected for the extracellular marker CD56 provide a populationof cells that are even more highly enriched in pancreatic progenitorcells than populations of pancreatic cells sorted by either markeralone. Surprisingly, in an animal model of diabetes, mice transplantedwith human pancreatic cells selected by the methods taught herein showedrestored normal levels of glucose. Accordingly, it is expected thatdiabetics can show improved control of glucose levels by transplantationwith pancreatic cells selected by the methods of the invention. Thus,the invention provides important new methods for helping diabeticindividuals reduce their dependence on exogenously administered insulin.

II. Definitions

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety. Terms not defined herein have their ordinary meaning asunderstood by a person of skill in the art.

“EphA4,” or “Ephrin type-A receptor 4” refers to a member of theephrin-A family with the EC number 2.7.1.112. It is a receptor tyrosinekinase, also known as “tyrosine-protein kinase receptor SEK” and“receptor protein-tyrosine kinase HEK8,” while the gene is known as“EPHA4,” “SEK,” “HEK8,” “TYRO1” or “LocusId:2043,” and maps onchromosome 2, at 2q36.1. The human form of the protein was originallyisolated from fetal brain tissue, as described in Fox et al., “cDNAcloning and tissue distribution of five human EPH-like receptorprotein-tyrosine kinases”, Oncogene 10:897-905 (1995). The sequence ofthe precursor form of the human protein is set forth in the Swiss-Protdatabase under accession number P54764 and can be found on the internetby entering “http://” followed by“us.expasy.org/cgi-bin/niceprot.pl?P54764”. EphA receptors bind toGPI-anchored ephrin-A ligands.

“CD56” is a cell surface protein, also known as Neural Cell AdhesionMolecule (N-CAM). CD56 is expressed on neurons, muscle cells, adrenalmedulla cells, astrocytes, Schwann cells, NK cells and a subset ofactivated T cells, including those that are β cell antigen-specific andknown to cause Type 1 diabetes. See e.g., Shliakhovenko et al., VrachDelo 2:453-459 (1991); Mechtersheimer et al., Ann. NY Acad. Sci.650:311-316 (1992); Leon et al., Brain Res. Dev. Brain Res. 70:109-121(1992); Pierre et al. Neuroscience 103:133-142 (2001); Hung et al. Glai38:363-370 (2002); and Ami et al., Clin. Exp Immunol. 128:453-459(2002). Previous work by one of the present inventors showed that CD56is also an extracellular marker for progenitors of pancreatic 13 cells.CD56 has a developmental role in pattern formation, by facilitatingcell-cell interactions. Known binding partners of CD 56 include otherCD56 proteins and heparin or heparin sulfate. The majority of CD56proteins are found in three isoforms resulting from differentialsplicing of mRNA: a 180 kD form, a 140 kD form, and a 120 kD form. CD56proteins are extensively post-translationally modified. Posttranslational modifications include addition of asparagine linkedoligosaccharides, sulfation of oligosaccharides, phosphorylation ofserine and threonine residues, and fatty acid acylation of the protein.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to CD56proteins, polymorphic variants, alleles, orthologs, and conservativelymodified variants, or splice variants, or portions thereof, can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with CD56 proteins and not with otherproteins. This selection may be achieved by subtracting out antibodiesthat cross-react with other molecules. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Specific binding canalso be used to describe the interaction of other molecules thatspecifically bind to CD56 protein, e.g. CD56 ligands and lectins thatrecognize CD56.

An “antigen” is a molecule that is recognized and bound by an antibody,e.g., peptides, carbohydrates, organic molecules, or more complexmolecules such as glycolipids and glycoproteins. The part of the antigenthat is the target of antibody binding is an antigenic determinant and asmall functional group that corresponds to a single antigenicdeterminant is called a hapten.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

As used herein, “insulin producing cells” refers to cells that secretedetectable amounts of insulin. “Insulin producing cells” can beindividual cells or collections of cells. One example of a collection of“insulin producing cells” is “insulin producing cell aggregates” e.g.,an organized collection of cells with a surrounding mantle of CK-19positive cells and an inner cell mass. “Aggregate” in the context ofcells refers to a three dimensional structure. “CK-19” is a 40 kD acidickeratin, cytokeratin 19. “Mantle” refers to an envelope of cellssurrounding in three dimensions the inner cell mass.

The term “contacting” is used herein interchangeably with the following:combined with, added to, mixed with, passed over, incubated with, flowedover, etc.

The term “lectin” refers to protein that recognize specific carbohydratemolecules. In a preferred embodiment the carbohydrate is all or part ofan oligosaccharide linked to a CD56 protein molecule.

A “ligand” is a molecule that is specifically bound by a protein. As anexample, heparin and heparin sulfate are bound by the CD56 molecule. Theterm also encompasses molecules that bind to a protein, for example, anantibody that specifically binds to a protein. In some instances theligand binds to a molecule that is covalently linked to a protein, forexample, a carbohydrate or an oligosaccharide.

The terms “heparan or heparin and heparin sulfate” are known to those ofskill in the art. Heparin and heparin sulfate are examples ofglycosaminoglycans.

The term “FACS” refers to fluorescence activated cell sorting, atechnique used to separate cells according to their content ofparticular molecules of interest. The molecule of interest can bespecific for a type of cell or for particular cell state. The moleculeof interest can be fluorescently labeled directly by binding to afluorescent dye, or by binding to a second molecule, which has beenfluorescently labeled, e.g., an antibody or lectin that has beenfluorescently labeled and that specifically binds to the molecule ofinterest. In a preferred embodiment, a fluorescently labeled EphA4specific antibody is used to separate EphA4-positive cells fromEphA4-negative cells.

The term “panning” refers to a method of selecting cells that bind to,as appropriate in context, a EphA4− or CD56− binding reagent. A flatsurface, e.g., a culture dish, is coated with the chosen bindingreagent. Pancreatic cells are added to the surface and allowed to bindto the binding reagent. The culture dishes are then washed, removing thecells that have not bound (that is, cells that are either EphA4− orCD56− negative, depending on the binding reagent used) from the dish. Ina preferred embodiment, an EphA4− specific antibody is used to coat aculture dish and “pan” for EphA4-positive cells in a population ofpancreatic cells.

“Differentiate” or “differentiation” refers to a process where cellsprogress from an undifferentiated state to a differentiated state orfrom an immature state to a mature state. For example, undifferentiatedpancreatic cells are able to proliferate and express characteristicsmarkers, like PDX-1. Mature or differentiated pancreatic cells do notproliferate and secrete high levels of pancreatic endocrine hormones.E.g., mature β-cells secrete insulin at high levels. Changes in cellinteraction and maturation occur as cells lose markers ofundifferentiated cells or gain markers of differentiated cells. Loss orgain of a single marker can indicate that a cell has “matured ordifferentiated.”

The term “differentiation factors” refers to a compound added topancreatic cells to enhance their differentiation to mature insulinproducing β cells. Exemplary differentiation factors include hepatocytegrowth factor, keratinocyte growth factor, exendin-4, basic fibroblastgrowth factor, insulin-like growth factor-I, nerve growth factor,epidermal growth factor and platelet-derived growth factor.

The term “providing pancreatic function to a mammal in need of suchfunction” refers to a method of producing pancreatic hormones within thebody of a mammal unable to produce such hormones on its own. In apreferred embodiment, insulin is produced in the body of a diabeticmammal. The pancreatic function is provided by implanting ortransplanting aggregates of insulin producing pancreatic cells, producedby the methods of this disclosure into the mammal. The number ofaggregates implanted is an amount sufficient to produce a measurableamount of insulin in the mammal. The insulin can be measured by Westernblotting or by other detection methods known to those of skill in theart, including assays for insulin function, such as maintenance of bloodglucose levels. Insulin can also be measured by detecting C-peptide inthe blood. In another preferred embodiment, the provision of pancreaticfunction is sufficient to decrease or eliminate the dependence of themammal on insulin produced outside the body.

“Encapsulation” refers to a process where cells are surrounded by abiocompatible acellular material, such as sodium alginate andpolylysine. Preferably small molecules, like sugars and low molecularweight proteins, can be taken up from or secreted into an environmentsurrounding the encapsulated cells. At the same time access to theencapsulated cells by larger molecules and immune cells is limited.

“Implanting” is the grafting or placement of the cells into a recipient.It includes encapsulated cells and non-encapsulated. The cells can beplaced subcutaneously, intramuscularly, intraportally orinterperitoneally by methods known in the art.

A “population” of cells refers to a plurality of cells obtained by aparticular isolation or culture procedure. While the selection processesof the present invention yield populations with relatively uniformproperties, a population of cells may be heterogeneous when assayed formarker expression or other phenotype. Properties of a cell populationare generally defined by a percentage of individual cells having theparticular property (e.g., the percentage of cells staining positive fora particular marker) or the bulk average value of the property whenmeasured over the entire population (e.g., the amount of mRNA in alysate made from a cell population).

“Passage” of cells usually refers to a transition of a seeded culturecontainer from a partially confluent state to a confluent state, atwhich point they are removed from the culture container and reseeded ina culture container at a lower density. However, cells may be passagedprior to reaching confluence. Passage typically results in expansion ofthe cell population as they grow to reach confluence. The expansion ofthe cell population depends on the initial seeding density but istypically a 1 to 10, 1 to 5, 1 to 3, or 1 to 2 fold expansion. Thus,passaging generally requires that the cells be capable of a plurality ofcell divisions in culture.

III. Isolation of EphA4 Positive Pancreatic Cells

Those of skill in the art will recognize that a variety of sources andmethods can be used to isolate EphA4 positive pancreatic cells.

A. Isolation of Pancreas from a Donor

Pancreatic cells isolated for subsequent culturing are obtained from oneor more donated pancreases. The methods described herein are notdependent on the age of the donated pancreas. Accordingly, pancreaticmaterial isolated from donors ranging in age from embryos to adults canbe used.

In another embodiment, pancreatic cells are isolated from a culturedsource. For example, cells prepared according to the microencapsulationmethod of U.S. Pat. No. 5,762,959 to Soon-Shiong, et al., entitled“Microencapsulation of cells,” can be harvested as a source of donorcells.

1. Isolation of Pancreatic Cells from Pancreas

Once a pancreas is harvested from a donor, it is typically processed toyield individual cells or small groups of cells for culturing using avariety of methods. One such method calls for the harvested pancreatictissue to be cleaned and prepared for enzymatic digestion. Enzymaticprocessing is used to digest the connective tissue so that theparenchyma of the harvested tissue is dissociated into smaller units ofpancreatic cellular material. The harvested pancreatic tissue is treatedwith one or more enzymes to separate pancreatic cellular material,substructures, and individual pancreatic cells from the overallstructure of the harvested organ. Collagenase, DNAse, Liberasepreparations (see U.S. Pat. Nos. 5,830,741 and 5,753,485) and otherenzymes are contemplated for use with the methods disclosed herein.

Isolated source material can be further processed to enrich for one ormore desired cell populations. However, unfractionated pancreatictissue, once dissociated for culture, can also be used directly in theculture methods of the invention without further separation, and willyield the intermediate cell population. In one embodiment the isolatedpancreatic cellular material is purified by centrifugation through adensity gradient (e.g., Nycodenz®, Ficoll®, or Percoll®). For examplethe gradient method described in U.S. Pat. No. 5,739,033, can be used asa means for enriching the processed pancreatic material in islets. Themixture of cells harvested from the donor source will typically beheterogeneous and thus contain α-cells, β-cells, δ-cells, ductal cells,acinar cells, facultative progenitor cells, and other pancreatic celltypes.

A typical purification procedure results in the separation of theisolated cellular material into a number of layers or interfaces.Typically, two interfaces are formed. The upper interface isislet-enriched and typically contains 10 to 100% islet cells insuspension. The second interface is typically a mixed population ofcells containing islets, acinar, and ductal cells. The bottom layer isthe pellet, which is formed at the bottom of the gradient. This layertypically contains primarily (>80%) acinar cells, some entrapped islets,and some ductal cells. Ductal tree components can be collectedseparately for further manipulation.

The cellular constituency of the fractions selected for furthermanipulation will vary depending on which fraction of the gradient isselected and the final results of each isolation. When islet cells arethe desired cell type, a suitably enriched population of islet cellswithin an isolated fraction will contain at least 10% to 100% isletcells. Other pancreatic cell types and concentrations can also beharvested following enrichment. For example, the culture methodsdescribed herein can be used with cells isolated from the secondinterface, from the pellet, or from other fractions, depending on thepurification gradient used.

In one embodiment, intermediate pancreatic cell cultures are generatedfrom the islet-enriched (upper) fraction. Additionally, however, themore heterogeneous second interface and the bottom layer fractions thattypically contain mixed cell populations of islets, acinar, and ductalcells or ductal tree components, acinar cells, and some entrapped isletcells, respectively, can also be used in culture. While both layerscontain cells capable of giving rise to the EphA4-positive populationdescribed herein, each layer may have particular advantages for use withthe disclosed methods.

B. Selection of EphA4 Positive Pancreatic Cells

Once a source of pancreatic cells have been chosen, EphA4 positive cellscan be selected and then separated from cells that do not express EphA4.Those of skill in the art will recognize that a variety of methods canbe used to select EphA4-positive cells and separate those cells fromEphA4-negative cells.

1. Detection of EphA4 Positive Cells Using Molecules that Bind EphA4

Those of skill in the art will recognize that there are many methods todetect EphA4 protein. For example, antibodies that bind specifically tothe EphA4 protein can be used to detect EphA4. Antibodies specific tothe EphA4 protein are known to those of skill in the art and arecommercially available from, for example, Abcam (Cambridge, UnitedKingdom), BD Biosciences Pharmingen (San Diego, Calif.), Gene Tex (SanAntonio, Tex.), Novus Biologicals (Littleton, Calif.) and Upstate GroupLLC (Charlottesville, Va.). In addition to antibodies, other moleculesthat bind specifically to EphA4 can be used to identify EphA4-positivecells.

Those of skill in the art will recognize that molecules that bindspecifically to EphA4 are particularly useful if they are labeled andthus able to be detected by some means. A “label” is a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. For example, useful labels include³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., ascommonly used in an ELISA), biotin, digoxigenin, or haptens and proteinswhich can be made detectable, e.g., by incorporating a radiolabel intothe peptide or used to detect antibodies specifically reactive with thepeptide.

2. FACS to Select EphA4 Positive Cells

Fluorescently labeled molecules that bind specifically to EphA4, mostcommonly antibodies, are used to select EphA4 positive cells inconjunction with a Fluorescence Activated Cell Sorter (“FACS”). Briefly,pancreatic cells are incubated with fluorescently-labeled antibody andafter the antibody binding, the cells are analyzed by FACS. The cellsorter passes single cells suspended in liquid through a fluorimeter.The amount of fluorescence is measured and cells with fluorescencelevels detectably higher than control, unlabeled, cells are selected aspositive cells.

FACS can also be used to physically separate cell populations based onmeasurement of fluorescence. The flowing cells are deflected byelectromagnetic fields whose strength and direction are varied accordingto the measured intensity of the fluorescence signal. LabeledEphA4-positive cells can be deflected into a separate container andthus, separated from unlabeled, EphA4-negative cells.

After pancreatic cells are isolated from pancreas, the cells are firstcultured for one to two passages and then labeled with a EphA4-specificantibody. The cells are then scanned using FACS to separateEphA4-positive from EphA4-negative cells. Up to 98% of the cells aredeemed negative for EphA4.

Many different fluorescent molecules are available for conjugation toantibodies, for example fluorescien or rhodamine. Those of skill areaware that in some instances more than one extracellular marker can bedetected by using different antibodies conjugated to fluorescentmolecules. FACS analysis can be done under conditions to identify morethan one extracellular marker of interest. In some embodiments,antibodies to EphA4 and antibodies to CD56 are chosen to “double select”for cells bearing these markers, as discussed elsewhere in thisdisclosure.

3. Affinity Adsorbing EphA4-Positive Cells Onto a Solid Support.

EphA4-positive cells can also be separated from EphA4-negative cells byusing EphA4-specific binding molecules attached to a solid support.Those of skill in the art will recognize that EphA4-specific antibodiescan be bound to a solid support through an antibody binding molecule,such as protein G or protein A or alternatively, can be conjugated to asolid support directly.

EphA4-positive cells can also be separated from EphA4-negative cellsthrough the technique of panning. Panning is done by coating a solidsurface with a EphA4-binding reagent and incubating pancreatic cells onthe surface for a suitable time under suitable conditions. A flatsurface, e.g., a culture dish, is coated with a EphA4-binding reagent.Pancreatic cells are added to the surface and allowed to bind to theEphA4-binding reagent. The culture dishes are then washed, removing theEphA4-negative cells from the dish. In a preferred embodiment, aEphA4-specific antibody is used to coat a culture dish and “pan” forEphA4-positive cells in a population of pancreatic cells.

In some embodiments, the solid support is a bead or particle which iscoated with anti-EphA4 antibodies. EphA4-positive cells then bind to thebead or particle and can be separated from the EphA4-negative cells byany of a number of methods, such as by washing the media containing thecells through a screen with openings sized to permit unbound(EphA4-negative) cells to pass through, while retaining beads orparticles to which EphA4-positive cells have bound. In a preferred groupof embodiments, the beads are magnetic. The use of magnetic beadsfacilitates the separation step.

IV. Cell Culture and Cultivation of EphA4-Positive Cells and TheirProgeny

A. General Cell Culture Procedures

Once the pancreatic cells are obtained and isolated, they are culturedunder conditions that select for propagation of the desiredEphA4-positive population, or in other embodiments, for thedifferentiation of more mature cell types. General cell culturemethodology may be found in Freshney, Culture of Animal Cells: A Manualof Basic Technique 4th ed., John Wiley & Sons (2000). Typically,pancreatic cells are cultured under conditions appropriate to othermammalian cells, e.g., in humidified incubators at 37° C. in anatmosphere of 5% CO₂. Cells may be cultured on a variety of substratesknown in the art, e.g., borosilicate glass tubes, bottles, dishes,cloning rings with negative surface charge, plastic tissue culturetubes, dishes, flasks, multi-well plates, containers with increasedgrowth surface area (GSA) or Esophageal Doppler Monitor (EDM) finish,flasks with multiple internal sheets to increase GSA, Fenwal bags, andother culture containers.

Once the pancreatic cellular material has been harvested and selectedfor culture, or once a population is confluent and is to be transferredto a new substrate, a population of cells is seeded to a suitable tissueculture container for cultivation. Seeding densities can have an effecton the viability of the pancreatic cells cultured using the disclosedmethods, and optimal seeding densities for a particular culturecondition may be determined empirically by seeding the cells at a rangeof different densities and monitoring the resulting cell survival andproliferation rate. A range of seeding densities has been shown to beeffective in producing hormone secreting cells in culture. Typically,cell concentrations range from about 10² to 10⁸ cells per 100 mm culturedish, e.g., 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ cells per 100 mmculture dish, although lower cell concentrations may be employed forcloning procedures. Cell concentration for other culture vessels may beadjusted by computing the relative substrate surface area and/or mediumgas exchange surface area for a different culture vessel. For example, atypical 100 mm culture dish has a substrate surface area of 55 squarecentimeters (see Freshney, supra), and a cell concentration of 10,000cells per dish corresponds to about 180 cells per square centimeter,while a cell concentration of 100,000 cells per dish corresponds toabout 1,800 cells per square centimeter. Cell concentration in terms ofculture vessel surface area may be related to cell concentration interms of media volume by using the appropriate media volume per culturesurface area (0.2-0.5 ml/cm² are typical ranges for static culture). Todetermine if a 10 fold expansion has occurred, the cells are removed byenzymatic digestion and counted under microscope in a known volume offluid. Cells may also be grown on culture surfaces pre-coated withdefined extracellular matrix components to encourage growth anddifferentiation (e.g., fibronectin, Collagen I, Engelbreth-Holm-Swarmmatrix, and, preferably, collagen IV or laminin).

Standard cell culture propagation techniques are suitable for practiceof the invention. When cells are growing attached to a culture surface,they are typically grown as a monolayer until 80%-90% confluence isreached, at which point the cells are released from the surface byproteolytic digestion and split 1:2 or 1:3 for culture in new vessels.Higher dilutions of the cells are also suitable, generally between theranges of 1:4 to 1:10, although even lower cell concentrations areappropriate in cloning procedures. Concentrations of proteolytic enzymesand chelating agents are usually lowered when cells are passaged inserum-free media (e.g., 0.025% trypsin and 0.53 mM EDTA). Culture mediumis typically changed twice weekly or when the pH of the medium indicatesthat fresh medium is needed.

The pancreatic cells of the present invention may be cultured in avariety of media. As described herein, media containing or lackingparticular components, especially serum, are preferred for certain stepsof the isolation and propagation procedures. For example, cells freshlyisolated from the pancreas may be maintained in high serum medium toallow the cells to recover from the isolation procedure. Conversely, lowserum medium favors the selection and propagation of an intermediatestage population. Accordingly, a number of media formulations are usefulin the practice of the invention. The media formulations disclosed hereare for exemplary purposes, and non-critical components of the media maybe omitted, substituted, varied, or added to simply by assaying theeffect of the variation on the replication or differentiation of thecell population, using the assays described herein. See, e.g., Stephanet al., Endocrinology 140:5841-54 (1999)).

Culture media usually comprise a basal medium, which includes inorganicsalts, buffers, amino acids, vitamins, an energy source, and, in somecases, additional nutrients in the form of organic intermediates andprecursors that are involved in protein, nucleic acid, carbohydrate, orlipid metabolism. Basal media include F12, Eagle's MEM, Dulbecco'smodified MEM (DMEM), RPMI 1640, a 1:1 mixture of F12 and DMEM, andothers. See Freshney, supra. To support the growth of cells, basalmedium is usually supplemented with a source of growth factors, otherproteins, hormones, and trace elements. These supplements encouragegrowth, maintenance, and/or differentiation of cells, compensate forimpurities or toxins in other medium components, and providemicronutrients lacking in the basal medium. In many culture media, serumis the source of these supplements. Serum can be supplied from a varietyof mammalian sources, such as human, bovine, ovine, equine, and thelike, and from adult, juvenile, or fetal sources. See Freshney, supra.Fetal bovine serum is a commonly used supplement. Concentrations ofserum are expressed in terms of volume of serum as a percentage of thetotal medium volume, and typically range from about 0.1 to 25%, e.g.,about 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25%. Insome applications, the basal medium is supplemented with defined orsemi-defined mixtures of growth factors, hormones, and micronutrients,rather than with serum. Formulas for serum replacement supplements aredisclosed herein; others are known in the art or available fromcommercial sources (see Freshney, supra). For some embodiments, theconcentration of serum is lowered but not eliminated, and defined orsemi-defined supplement mixtures are added to the basal medium.Preferred applications for media containing high or low concentrationsof serum are described herein.

B. Maintenance and Propagation of Isolated Pancreatic Cells in MediaContaining High Serum

Cells harvested from a donor pancreas have usually undergone a period ofwarm or cold ischemia between the death of the donor and the beginningof the isolation procedure. Moreover, during the isolation procedure,pancreatic cells are usually subjected to proteolytic digestion as wellas mechanical and shear stresses. Without wishing to be bound by aparticular theory, the various traumas experienced by these cells mayup-regulate various cellular processes that result in the expansion ofpancreatic stem cell populations, such as facultative progenitor cells.Intermediate cell populations may be generated with satisfactoryefficiency by placing cells into low serum media directly afterisolation or purification. Nonetheless, because the trauma experiencedby cells during the isolation procedures may have adverse effects oncell survival and adaptation to culture, it is sometimes desirable tomaintain the freshly isolated cells in a stabilizing medium containinghigh concentrations of serum (e.g., >4%) to improve the efficiency ofthe culturing process. This maintenance period may be brief (e.g.,overnight). Optionally, cells may be maintained for an extendedpropagation period in high serum medium.

High serum media for stabilization will typically contain at least 4%serum, and, in some embodiments, will contain a higher concentration ofserum such as 10% or 20%. Media used for stabilization or propagationmay be derived from a basal medium such as RPMI 1640, available frommany commercial sources and described by Moore et al., J Am Med Assoc199:519-524 (1967)). Exemplary high serum media for maintenance orpropagation include Medium 3 (RPMI 1640+10 mM HEPES, 2 mM glutamine, 5μM ZnSO₄, and 10% fetal bovine serum (FBS)) and Medium 7 (RPMI 1640+10mM HEPES, 2 mM glutamine, 5 μM ZnSO₄, and 20% FBS). High serum media mayalso be derived by mixing a particular volume of high serum medium suchas Medium 3 or Medium 7 with a particular volume of serum-free mediumsuch as SM95, SM96, or SM98 (described herein) to arrive at a desiredserum concentration (e.g., 4%-9%).

For stabilization after harvest, cells are conveniently cultured in aculture vessel at relatively high densities in a high serum medium(e.g., 10⁹ cells in 70 ml of Medium 7 (20% FBS)). However, lower celldensities and serum concentrations may be employed as well. Cells aretypically maintained in the original vessel for a relatively short time(e.g., overnight) to allow for recovery from the harvesting procedure.

Following the maintenance period, cells may be transferred to low serummedia for selection and propagation of the EphA4-positive cellpopulation as described herein. Optionally, the cells may be cultured ina high serum medium to allow for proliferation of the mixed cellpopulation. In a typical embodiment, cells from the maintenance cultureare reseeded into a new culture vessel containing Medium 3 (10% FBS),Medium 7 (20% FBS), or a mixture of Medium 3 and Medium 7 (15% FBS), orother AmCyte culture media. Cells are typically cultured in this mediumfor 7-10 days, during which time they may grow to confluence. Once thecells have reached confluence, they may be passaged into low serum mediafor selective expansion of the intermediate cell population describedherein.

C. Expansion and Propagation of a EphA4 Positive Pancreatic CellPopulation by Culture in Media Containing Low Serum

Once the pancreatic cells have been isolated, the cells are thentransferred to a selective medium to promote the emergence of apropagating intermediate stage population. This selective medium favorspropagation of cells which retain the ability to secrete pancreaticendocrine hormones, or which retain the potential to mature into moredifferentiated cells which secrete high levels of pancreatic endocrinehormones. In general, selective medium will favor propagation ofepithelial or epithelial-like cells at the expense of fibroblasts andmesenchymal cells, although pure epithelial cultures have not been shownto be required for the advantageous use of pancreatic cells in themethods of the invention. Typically, epithelial-selective media willyield a population of nearly pure (e.g., <10% fibroblasts or mesenchymalcells) cells after a certain period of growth in culture, e.g., 2, 3, 4,or 5 passages depending on the expansion of the population in eachpassage.

One type of selective medium which has been employed to favor epithelialcell growth from embryonic tissues is serum-free medium (see, e.g.,Stephan et al., supra; Peehl and Ham, In Vitro 16:526-40 (1980)).Epithelial-specific media, and, more preferably, low serum mediacontaining a source of growth hormone, may be employed to select for adistinct population of propagating pancreatic cells from adult mammalsthat retain markers of pancreatic cell development (e.g., PDX-1), butcan be further differentiated under appropriate conditions to expresshigh levels of pancreatic endocrine hormones. Particularepithelial-selective media suitable for culture of pancreatic cells aredisclosed herein, but other medium formulations known in the art tofavor the preferential expansion of epithelial or epithelial-like cellsmay also be employed.

The transfer to epithelial-selective low serum medium may beaccomplished after a period of maintenance in high serum medium(“weaning”), or by transferring the cells directly into selective lowserum medium following the isolation and separation procedure (“shock”).Either methodology is suitable for generation of the desiredintermediate cell population.

1. Supplements

Typical ingredients added to basal media for complete serum-free mediainclude recombinant human insulin (0.1 to 100 μg/ml), transferrin (0.1to 100 μg/ml), epidermal growth factor (0.1 to 100 ng/ml), ethanolamine(0.1 to 100 μg/ml), aprotinin (0.1 to 100 μg/ml), glucose (0.1 to 100mg/ml), phosphoethanolamine (0.1 to 100 μM), triiodothyronone (0.1 to100 pM), selenium (0.1 to 100 nM), hydrocortisone (0.01 to 100 μM),progesterone (0.1 to 10 nM), forskolin (0.1 to 100 μM), heregulin (0.1to 100 nM), and bovine pituitary extract (0.1 to 500 μg/ml). Not allsupplemental ingredients are required to support cell growth; theoptimal concentration or necessity for a particular supplement may bedetermined empirically, by leaving out or reducing the concentration ofa single ingredient and observing the effect on cell proliferation. Seee.g., Stephan et al., supra.

In general, supplemental ingredients may be replaced by natural orsynthetic products that have the same biological properties. Forexample, triiodothyronone, hydrocortisone, and progesterone may all bereplaced by natural or synthetic hormones known to activate the sameintracellular receptors (thyroid receptors, glucocorticoid receptors,and progesterone receptors). Insulin and EGF are typically humanproteins produced by recombinant DNA methodology, but may be replaced bypolypeptides purified from natural sources, by polypeptides from otherspecies, or by other agonists of the insulin and EGF receptors. Growthhormone (“GH”, mature human growth hormone is a 191-amino acid peptidewhich displays a molecular mass of 22 kD) may be used or, in some cases,may be substituted with other agents which bind to the GH receptor.Likewise, heregulin, a ligand of the ErbB3 receptor, may be replaced byheregulin isoforms and other ErbB3 agonists such as NRG2, NRG3, andNRG4, sensory and motor neuron-derived factor, neurestin, and Ebp-1,heregulin α, heregulin β, heregulin γ, neuregulin-1 and neuregulin-2(NRG-1 alpha, NRG-1beta, NRG-2 alpha, and NRG-2 beta.

Exemplary serum-free media include the basal medium SM96 and thecomplete medium SM95, which consists of SM96 supplemented as shown inthe following tables. SM98 consists of 1:1 F12/DMEM supplemented with amodification of medium supplement 14F described by Stephan et al.,supra. SM98 contains less heregulin (1 ng/ml v. 8 ng/ml) than 14F. Thus,SM 98 consists of 1:1 F12/DMEM supplemented with recombinant humaninsulin, 10 μg/ml; transferrin, 10 μg/ml; epidermal growth factor, 10ng/ml; ethanolamine, 61 ng/ml; aprotinin, 25 μg/ml; glucose, 5 mg/ml;phosphoethanolamine, 141 ng/ml; triiodothyronone, 3.365 pg/ml; selenium,4.325 ng/ml; hydrocortisone, 181 ng/ml; progesterone, 3.15 ng/ml;forskolin, 410 ng/ml; heregulin, 1 ng/ml; and bovine pituitary extract,75 μg/ml. Exemplary sources of EGH and heregulin in SM95 and SM98 arerecombinant human EGF (Sigma-Aldrich Co., St. Louis, Mo., catalog numberE9644) and the EGF domain (amino acids 176-246) of human heregulin-β1(R&D Systems Inc., Minneapolis, Minn., catalog number 396-HB/CF).

RPMI 1640 Media (Moore, et al., A.M.A., 199: 519 (1967)) Mg/L INORGANICSALTS Ca(NO₃)₂—4H₂O 100 KCl 400.00 MgSO₄ (anhyd.) 48.84 NaCl 5850.00Na₂HPO₄ (anhyd.) 800.00 OTHER COMPONENTS D-Glucose 2000.00 Glutathione(reduced) 1.0 HEPES 5958.00 Phenol Red 5.00 AMINO ACIDS L-Arginine200.00 L-Asparagine (free base) 50.00 L-Aspartic Acid 20.00L-Cystine•2HCl 65.00 L-Glutamic Acid 20.00 L-Glutamine 300.00 Glycine10.00 L-Histidine (free base) 15.00 L-Isoleucine 50.00 L-Leucine 50.00AMINO ACIDS L-Lysine•HCl 40.00 L-Methionine 15.00 L-Phenylalanine 15.00L-Proline 20.00 L-Serine 30.00 L-Threonine 20.00 L-Tryptophan 5.00L-Tyrosine•2Na₂H₂0 29.00 L-Valine 20.00 VITAMINS Biotin 0.20 D-CaPantothenate 0.25 Choline Chloride 3.00 Folic Acid 1.00 i-Inositol 35.00Niacinamide 1.00 Pyridoxine•HCl 1.00 Riboflavin 0.20 Thiamine•HCl 1.00Thymidine 0.005 Vitamin B₁₂ 1.04

SM95 Mg/L INORGANIC SALTS CaC1₂ 78.3 CuS0₄•5H₂0 0.00165 Fe(NO₃)₃•9H₂O0.025 FeSO₄•7H₂0 0.61 KCl 271 MgC1₂ 28.36 MgSO₄ 39.06 KH₂PO₄ 34 NaCl7262.75 NaHCO₃ 1600 Na₂HPO₄ 101.5 NaH₂PO₄•H₂O 31.25 ZnS0₄•7H₂O 0.416AMINO ACIDS L-Alanine 11.225 L-Arginine•HCl 283.75 L-Asparagine•H₂018.75 L-Aspartic Acid 16.325 L-Cysteine•H₂0(non-animal) 43.78L-Cystine•2HCl 15.65 L-Glutamic Acid 18.675 L-Glutamax I 328.5 Glycine89.375 Glycyl-Histidyl-Lysine 0.000005 L-Histidine HC1•H₂0 38.69L-Isoleucine 31.24 L-Leucine 42.5 L-Lysine•HCl 82.125 L-Methionine 13.12L-Phenylalanine 22.74 L-Proline 43.625 L-Serine 23.625 L-Threonine38.726 L-Tryptophan 6.51 L-Tyrosine•2Na₂H₂0 (non-animal) 35.9 L-Valine38.125 OTHER COMPONENTS D-Glucose 3000 HEPES 1787.25 Na Hypoxanthine 3.2Linoleic Acid 0.066 Lipoic Acid 0.1525 Phenol Red 4.675 NaPutrescine•2HCl 0.191 Na Pyruvate 137.5 VITAMINS Biotin 0.037 AscorbicAcid 22.5 D-Ca Pantothenate 1.37 Choline Chloride 11.49 Folic Acid 1.826L-Inositol 24.3 Niacinamide 1.03 Pyridoxine•HCl 1.046 Riboflavin 0.13Thiamine•HCl 1.23 Thymidine 0.5325 Vitamin B₁₂ 1.04 SUPPLEMENTS NaSelenous Acid 0.0034 Epithelial Growth Factor 0.005 Ethanolamine 0.03Phosphoethanolamine 0.07 Aprotinin 12.5 Progesterone 0.0016 Forskolin0.205 HeregulinB 0.004 Bovine Pituitary Extract 37.5 Hydrocortisone0.0923 r.h. insulin 5.05 T₃ 0.0000015 L-Thyroxine Na 0.00002 BovineTransferrin APG 7.5

SM96 Mg/L INORGANIC SALTS CaC1₂ 78.3 CuS0₄•5H₂0 0.00165 Fe(NO₃)₃•9H₂O0.025 FeSO₄•7H₂0 0.61 KCl 271 MgC1₂ 28.36 MgSO₄ 39.06 KH₂PO₄ 34 NaCl7262.75 NaHCO₃ 1600 Na₂HPO₄ 101.5 NaH₂PO₄•H₂O 31.25 ZnS0₄•7H₂O 0.416AMINO ACIDS L-Alanine 11.225 L-Arginine•HCl 283.75 L-Asparagine•H₂018.75 L-Aspartic Acid 16.325 L-Cysteine•H₂0(non-animal) 43.78L-Cystine•2HCl 15.65 L-Glutamic Acid 18.675 L-Glutamax I 328.5 Glycine89.375 Glycyl-Histidyl-Lysine 0.000005 L-Histidine HC1•H₂0 38.69L-Isoleucine 31.24 L-Leucine 42.5 L-Lysine•HCl 82.125 L-Methionine 13.12L-Phenylalanine 22.74 L-Proline 43.625 L-Serine 23.625 L-Threonine38.726 L-Tryptophan 6.51 L-Tyrosine•2Na₂H₂0 (non-animal) 35.9 L-Valine38.1261 OTHER COMPONENTS D-Glucose 3000 HEPES 1787.25 Na Hypoxanthine3.2 Linoleic Acid 0.066 Lipoic Acid 0.1525 Phenol Red 4.675 NaPutrescine•2HCl 0.191 Na Pyruvate 137.5 VITAMINS Biotin 0.037 AscorbicAcid 22.5 D-Ca Pantothenate 1.37 Choline Chloride 11.49 Folic Acid 1.826i-Inositol 24.3 Niacinamide 1.03 Pyridoxine•HCl 1.046 Riboflavin 0.13Thiamine•HCl 1.23 Thymidine 0.6325 Vitamin B₁₂ 1.04

2. Transfer of Cells to Low Serum Media

Transferring a culture of pancreatic cells to low serum media promotesthe selection of a defined population of cells with an intermediatestate of differentiation. This cell population will continue toproliferate if subcultured, but maintains high expression levels ofpancreatic markers such as PDX-1. Unstimulated, this population secretesrelatively low levels of pancreatic endocrine hormones such as insulin,but can be matured according to the methods of the invention to yieldhigh-secreting cells. To transfer a culture of pancreatic cells to lowserum medium, the cells may be weaned from high serum to low serummedia, or may be placed directly in low serum media following isolation.Medium such as SM95 and SM98 are suitable low serum media, although SM95yields slightly improved insulin secretion upon maturation the ofpancreatic cells.

The EphA4-positive cell population and its progeny typically retainsboth the ability to proliferate and the ability for furtherdifferentiation into high-secreting endocrine cells. As theEphA4-positive cells proliferate, the strength of EphA4 expression canbecome less pronounced, and may be detectable only by RT-PCR.

The ability of EphA4 cells to proliferate provides an advantage in theirability to expand and increase the number of cells available for latermaturation into glucose-secreting, insulin-producing aggregates.Proliferative ability is generally assessed by the ability of a cultureseeded at a one density to expand to a second density; e.g., cellsplated at 180 cells per square centimeter may be expanded to 1,800 cellsper ml in a single passage. By repeated cycles of propagation andpassage, a starting population of isolated pancreatic cells may beexpanded by about 10,000-fold or more (e.g., about 100-fold, 500-fold,1000-fold, 5000-fold, 10,000-fold, 50,000-fold, 100,000-fold,500,000-fold, or 1,000,000 fold) while retaining endocrine markers suchas PDX-1 and insulin mRNA expression, and retaining the ability todifferentiate into mature high-secreting endocrine cells.

V. Differentiation-Induction of Insulin Producing Aggregates

Cell differentiation of EphA4-positive cells can be induced throughinduction of cell aggregation. As the EphA4-positive cellsdifferentiate, the strength of EphA4 expression can become lesspronounced. Cell aggregation can be induced in a variety of ways. Forexample, aggregation and differentiation can be induced by growing thecells to confluence. Aggregation and differentiation can also be inducedby growing cells on conditioned culture dishes.

A variety of substrates can be used to condition culture dishes.Conditioned culture dishes can be culture dishes that have been usedpreviously to grow intermediate stage pancreatic stem cells. Once thecells have formed a monolayer (typically about 5 days, depending on theinitial subculture seeding density), they are removed by trypsinization.Growth of a 100% confluent cell culture is not required to produce aconditioned culture dish. A lowered concentration of trypsin (typically½ or ¼ of the concentration employed in standard cell culturetechniques) is preferred to prevent extensive degradation of the matrix.Alternatively, the cell monolayer may be removed by extracting thesubstrate with detergent, which will remove the cells but leave behindthe secreted matrix (see Gospodarowicz et al., Proc Natl Acad Sci USA77:4094-8 (1980)).

Conveniently, the removed cells which previously grew on the substrateor culture dish may be split and reseeded on the same, now conditioned,culture dish. However, the culture which conditions the substrate andthe culture which is seeded on the substrate need not be the sameculture. Accordingly, one culture of cells may be grown on a substrateto condition the substrate, the cells removed, and cells from anotherculture seeded upon the conditioned substrate. The conditioning cellsmay be from the same or different donor or species as the cellssubsequently cultured.

In another embodiment, plates conditioned with collagen coating are usedin the invention. Collagen coated plates are commercially available. Ina preferred embodiment, collagen IV coated plates are used to induceaggregation and differentiation of pancreatic cells.

Differentiation of EphA4-positive cells into mature insulin producingcells can also be enhanced by growth of the cells in the presence ofdifferentiation factors. Preferred differentiation factors includehepatocyte growth factor, keratinocyte growth factor, and exendin-4.Hepatocyte growth factor has been shown to effect differentiation ofpancreatic cells in culture and in transgenic animals. See e.g.,Mashima, H. et al., Endocrinology, 137:3969-3976 (1996); Garcia-Ocana,A. et al., J. Biol. Chem. 275:1226-1232 (2000); and Gahr, S. et al., J.Mol. Endocrinol. 28:99-110 (2002). Keratinocyte growth factor has beenshown to effect differentiation of pancreatic cells in transgenicanimals. See e.g., Krakowski, M. L., et al., Am. J. Path. 154:683-691(1999) and Krakowski, M. L., et al., J. Endochrinol. 162:167-175 (1999).Exendin-4 has been shown to effect differentiation of pancreatic cellsin culture. See e.g., Doyle M. E. and Egan J. M., Recent Prog. Horm.Res. 56:377-399 (2001) and Goke, R., et al., J. Biol. Chem.268:19650-19655 (1993). bFGF has been shown to increase the insulinsecretion in microencapsulated pancreatic islets. See e.g., Wang W., etal., Cell Transplant 10(4-5): 465-471 (2001). IGF-I has an effect ondifferentiation of pancreatic ductal cells and IGF-I replacement therapyhas been used for type I diabetes treatment. See e.g., Smith F E., etal., Proc. Natl. Acad. Sci. USA. 15; 88(14): 6152-6156 (1991),Thrailkill K M. et al., Diabetes Technol. Ther. 2(1): 69-80 (2000).Evidence has shown that NGF plays an important autoregulatory role inpancreatic beta-cell function. See e.g. Rosenbaum T. et al., Diabetes50(8): 1755-1762 (2001), Vidaltamayo R. et al., FASEB 16(8): 891-892(2002), and Pierucci D. et al., Diabetologia 44(10): 1281-1295 (2001).EGF has been shown to promote islet growth and stimulate insulinsecretion. See e.g., Chatterjee A K. et al., Horm. Metab. Res. 18(12):873-874 (1986).

VI. Characterization of EphA4-Positive Cells and Their Progeny

Those of skill in the art will recognize that it can be useful todetermine the differentiation state of EphA4-positive cells and theirprogeny. The differentiation state of pancreatic cells can be determinedin a variety of ways, including measurement of protein and mRNA markersof differentiation and functional assays of pancreatic cells, e.g.ability to secrete insulin in response to glucose stimulation.

A. Phenotypic Assays

To know when mature pancreatic cells are present, it is useful to assaythe phenotypes of pancreatic cells at particular stages of culture.Since expression of particular proteins correlates with cell identity ordifferentiation state, cells may be analyzed for the expression of amarker gene or protein to assess their identity or differentiationstate. For example, in freshly isolated pancreatic tissue, expression ofamylase identifies the cell as an exocrine acinar cell, while expressionof insulin identifies the cell as an endocrine islet cell. Likewise,islet cells at an early stage of differentiation are usually positivefor the cytokeratin CK-19, while mature islet cells show less expressionof CK-19.

Phenotypic properties may be assayed on a cell-by-cell basis or as apopulation average. The mode of assay will depend on the particularrequirements and methodology of the assay technique. Thus, assays ofmarker expression by immunohistochemistry, performed on fixed sectionsor on suspended cells by FACS analysis, measure the frequency andintensity with which individual cells express a given marker. On theother hand, it may be desirable to measure properties such as theaverage insulin to actin mRNA expression ratio over an entire populationof cells. In such cases, the assay is typically performed by collectingmRNA from a pool of cells and measuring the total abundance of insulinand actin messages. Many phenotypic properties may be assayed either ona cell or population basis. For example, insulin expression may beassayed either by staining individual cells for the presence of insulinin secretory granules, or by lysing a pool of cells and assaying fortotal insulin protein. Similarly, mRNA abundance may be measured over apopulation of cells by lysing the cells and collecting the mRNA, or onan individual cell basis by in situ hybridization.

1. Cell Differentiation Markers

There are a number of cellular markers that can be used to identifypopulations of pancreatic cells. Donor cells isolated and cultured beginto display various phenotypic and genotypic indicia of differentiatedpancreatic cells. Examples of the phenotypic and genotypic indiciainclude various molecular markers present in the facultative progenitorcell population that are modulated (e.g., either up or down regulated).These molecular markers include CK-19, which is hypothesized to be amarker of the pancreatic facultative stem cell.

Typically, mammalian stem cells proceed through a number ofdevelopmental stages as they mature to their ultimate developmentalendpoint. Developmental stages often can be determined by identifyingmarkers present or absent in developing cells. Because human endocrinecells develop in a similar manner, various markers can be used toidentify cells as they transition from a stem cell-like phenotype topseudoislet phenotype.

The expression of markers in cells induced to proliferate ordifferentiate by the methods of the present invention bears somesimilarity to the sequence of marker expression in normal human pancreasdevelopment. Very early in development, the primordial epithelial cellsexpress PDX-1, an early cellular marker that is a homeodomain nuclearfactor. As the cells develop, they begin to bud out and form a duct.These cells express cytokeratin 19, a marker for epithelial ductalcells, and temporally express PDX-1 leading developmentally to endocrinecells. As these cells continue to develop, they gain the ability toexpress insulin, somatostatin, or glucagon. The final differentiatedcells are only able to express one and become the α cells (glucagon), βcells (insulin), and δ cells (somatostatin). The EphA4-positive cellpopulation used herein is believed to be at a less than fullydifferentiated stage of development, retaining the ability toproliferate and the potential to differentiate into mature endocrinecells. Whether the cells are indeed examples of a precursor in thedevelopment pathway or simply a result of in vitro manipulation, theEphA4-positive cells are able to proliferate as well as to expressendocrine hormones and, therefore, have the potential for being used tocorrect a deficiency in any type of islet cell.

Markers of interest are molecules that are expressed in temporal- andtissue-specific patterns in the pancreas (see Hollingsworth, Ann N YAcad Sci 880:38-49 (1999)). These molecular markers are divided intothree general categories: transcription factors, notch pathway markers,and intermediate filament markers. Examples of transcription factormarkers include PDX-1, NeuroD, Nkx-6.1, Isl-1, Pax-6, Pax-4, Ngn-3, andHES-1. Examples of notch pathway markers include Notch1, Notch2, Notch3,Notch4, Jagged1, Jagged2, Dll1, and RBPjk. Examples of intermediatefilament markers include CK19 and nestin. Examples of markers ofprecursors of pancreatic β cells include PDX-1, Pax-4, Ngn-3, and Hb9.Examples of markers of mature pancreatic β cells include insulin,somatostatin, glp-9, and glucagon.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art and includequantitative reverse transcription polymerase chain reaction (RT-PCR),Northern blots, and in situ hybridization (see, e.g., Current Protocolsin Molecular Biology (Ausubel et al., eds. 2001 supplement)) andimmunoassays, such as immunohistochemical analysis of sectionedmaterial, Western blotting, and, for markers that are accessible inintact cells, flow cytometry analysis (FACS) (see, e.g., Harlow andLane, Using Antibodies: A Laboratory Manual, New York: Cold SpringHarbor Laboratory Press (1998)). Conventional histochemical markers ofendocrine cell differentiation may also be employed. Cells to beexamined by immunohistochemistry may be cultured on glass chamber slidesfor microscopic examination. Alternatively, cells grown in conventionaltissue culture may be manually removed from the culture and embedded inparaffin for sectioning. PDX-1 antibody can be made following theteachings of Leonard J. et al., Mol. Endocrinol., 1993, Oct. 7, (10)1275-83.

Cell differentiation markers are varied and can be detected byconventional immunohistochemistry. A generally applicable protocolfollows.

The staining process begins with removing chamber portion of the slides.Cells were very gently rinsed with in buffers and fixed inparaformaldehyde solution. Cells are then incubated in a blockingsolution containing normal serum at room temperature. Cells werepermeabilized with non-ionic detergent in blocking solution. Primaryantibodies as listed below are prepared in blocking solution atappropriate dilution and added to cells and incubated. Followingincubating with primary antibody, cells were rinsed in buffer andreblocked in blocking solution.

Secondary antibody prepared in blocking solution at appropriate dilutionis added to the cells and incubated in the dark. Following incubationthe cells are rinsed and nuclei were counterstained with Hoechst dye.Excess fluid is removed and the slides are mounted and covered withcoverslides. The slides dry and are stored in the dark.

Alternatively the cells can be prepared for immunocytochemistry usingthe ABC method. In brief, the cells are embedded in parafin and slideswith paraffin sections are dried at 37° C. overnight. The cells aredeparaffinized and immersed in a hydrogen peroxide methanol solution toinhibit endogenous peroxidase activity. Slides were boiled in 0.01citrate buffer (pH 6.0) for 30 minutes to recover certain epitopes.Slides were rinsed with buffer and blocked using normal serum at roomtemperature in a moist chamber.

Primary antibody prepared in blocking solution are added to the samplesand incubated in a moist chamber. Slides are washed and incubated withsecondary antibody prepared in blocking solution. Slides were againrinsed with buffer and incubated with Avidin-Horse Reddish Peroxidesreagent or ABC complex from a commercial kit (e.g. Dako Corporation).Slides are again rinsed and incubated with diaminobenzidin developingsolution; urea hydrogen peroxides in a gold wrap. After washes withdistilled water, slides are immersed in Mayer's Hematoxylin for 5minutes, then kept slides in running tap water until water turnedcolorless and nuclei were blue. Slides are dehydrated and mounted forviewing.

2. Insulin mRNA Expression

One marker that may be used to characterize pancreatic cell identity,differentiation, or maturity is the level of insulin mRNA. For example,the intermediate cell population of the present invention showexpression of insulin mRNA within a defined range. Method forquantitating insulin mRNA include Northern blots, nuclease protection,and primer extension. In one embodiment, RNA is extracted from apopulation of cultured cells, and the amount of proinsulin message ismeasured by quantitative reverse transcription PCR. Following reversetranscription, insulin cDNA is specifically and quantitatively amplifiedfrom the sample using primers hybridizing to the insulin cDNA sequence,and amplification conditions under which the amount of amplified productis related to the amount of mRNA present in the sample (see, e.g., Zhouet al., J Biol Chem 272:25648-51 (1997)). Kinetic quantificationprocedures are preferred due to the accuracy with which starting mRNAlevels can be determined.

Frequently, the amount of insulin mRNA is normalized to a constitutivelyexpressed mRNA such as actin, which is specifically amplified from thesame RNA sample using actin-specific primers. Thus, the level ofexpression of insulin mRNA may be reported as the ratio of insulin mRNAamplification products to actin mRNA amplification products, or simplythe insulin:actin mRNA ratio. The expression of mRNAs encoding otherpancreatic hormones (e.g., somatostatin or glucagon) may be quantitatedby the same method. Insulin and actin mRNA levels can also be determinedby in situ hybridization and then used to determine insulin:actin mRNAratios. In situ hybridization methods are known to those of skill in theart.

B. Functional Assays

One of the important functions of a beta cell is to adjust its insulinsecretion according to the glucose level. Typically, a static glucosestimulation (SGS) assay can be performed on the proliferating adherentpancreatic cells to identify whether they are able to secrete insulin inresponse to different glucose levels. Cells are generally cultured on anappropriate substrate until nearly confluent. Three days prior to theSGS test, the culture medium is replaced by a medium of similarcharacter but lacking insulin and containing only 1 g/L of glucose. Themedium is changed each day for three days and the SGS test is performedon day four.

Before the test, the culture medium may be collected for glucose andinsulin analysis. To prepare cells for the test, cells are washed twicewith Dulbecco's phosphate-buffered saline (DPBS)+0.5% BSA, incubatingfor 5 minutes with each wash, and then once with DPBS alone, alsoincubating for 5 minutes. After washing, the cells are incubated with 10ml (in a 100 mm dish) or 5 ml (in a 60 mm dish) of Krebs-Ringers SGSsolution with 60 mg/dl glucose (KRB-60) for 30 minutes in a 37° C.incubator. This incubation is then repeated.

To perform the SGS assays, cells are incubated in 3 ml (100 mm dish) or4 ml (T75 flask) or 2 ml (60 mm dish) KRB-60, at 37° C. for 20 minutes.The medium is aspirated and spun, and is collected for insulin assay asLG-1 (low glucose stimulated step). KRB-450+theo (KRB with 450 mg/dlglucose and 10 mM theophylline) is then added with the same volume asabove, and cells are cultured under the same condition as above. Thesupernatant is collected for insulin assay as HG (high glucosestimulated). The cells are then incubated again with KRB-60 and themedium collected as LG-2, and another time as LG-3. The media arecollected for insulin analysis, and stored at −20° C. until insulincontent is determined by radioimmunoassay (RIA) or other suitable assay.

The results of the SGS test are often expressed as a stimulation index,defined as the HG insulin value divided by the LG-1 insulin value.Generally, a stimulation index of about 2 or greater is considered to bea positive result in the SGS assay, although other values (e.g., 1.5,2.5, 3.0, 3.5, etc.) may be used to define particular cell populations.

C. Preparation of EphA4-Positive Cells or Their Progeny for Implantationand Restoration of Pancreatic Endocrine Function

Those of skill in the art will recognize that propagating EphA4-positivecells provide a renewable resource for implantation and restoration ofpancreatic function in a mammal. Propagating EphA4-positive pancreaticcells are first differentiated before implantation into the mammal. Ifdesired by the user, EphA4 cells can be encapsulated beforeimplantation.

D. Encapsulation

Encapsulation of the EphA4-positive cells results in the formation ofcellular aggregates in the capsules. Encapsulation can allow thepancreatic cells to be transplanted into a diabetic host, whileminimizing the immune response of the host animal. The porosity of theencapsulation membrane can be selected to allow secretion ofbiomaterials, like insulin, from the capsule, while limiting access ofthe host's immune system to the foreign cells.

Encapsulation methods are known in the art and are disclosed in, forexample, the following references: van Schelfgaarde & de Vos, J. Mol.Med. 77:199-205 (1999), Uludag et al. Adv. Drug Del Rev. 42:29-64 (2000)and U.S. Pat. Nos. 5,762,959, 5,550,178, and 5,578,314. Encapsulationmethods are also described in detail in international applicationPCT/US02/41616; incorporated herein by reference.

E. Implantation

Implantation or transplantation into a mammal and subsequent monitoringof endocrine function may be carried out according to methods commonlyemployed for islet transplantation; see, e.g., Ryan et al., Diabetes50:710-19 (2001); Peck et al., Ann Med 33:186-92 (2001); Shapiro et al.,N Engl J Med 343(4):230-8 (2000); Carlsson et al., Ups J Med Sci105(2):107-23 (2000) and Kuhtreiber, W M, Cell Encapsulation Technologyand Therapeutics, Birkhauser, Boston, 1999. Preferred sites ofimplantation include the peritoneal cavity, the liver, and the kidneycapsule.

One of skill in the art will be able to determine an appropriate dosageof microcapsules for an intended recipient. The dosage will depend onthe insulin requirements of the recipient. Insulin levels secreted bythe microcapsules can be determined immunologically or by amount ofbiological activity. The recipients body weight can also be taken intoaccount when determining the dosage. If necessary, more than oneimplantation can be performed as the recipient's response to theencapsulated cells is monitored. Thus, the response to implantation canbe used as a guide for the dosage of encapsulated cells. (Ryan et al.,Diabetes 50:710-19 (2001))

F. In Vivo Measure of Pancreatic Endocrine Function

The function of encapsulated cells in a recipient can be determined bymonitoring the response of the recipient to glucose. Implantation of theencapsulated cells can result in control of blood glucose levels. Inaddition, evidence of increased levels of pancreatic endocrine hormones,insulin, C-peptide, glucagon, and somatostatin can indicate function ofthe transplanted encapsulated cells.

One of skill in the art will recognize that control of blood glucose canbe monitored in different ways. For example, blood glucose can bemeasured directly, as can body weight and insulin requirements. Oralglucose tolerance tests can also be given. Renal function can also bedetermined as can other metabolic parameters. (Soon-Shiong, P. et al.,PNAS USA 90:5843-5847 (1993); Soon-Shiong, P. et al., Lancet 343:950-951(1994)).

VII. Double-Selection of EphA4− and CD56− Positive Cells

In some embodiments, it is desirable to select for cells that expressboth EphA4 and CD56. CD56, also known as Neural Cell Adhesion Molecule(N-CAM), is a cell surface protein. International applicationPCT/US2003/028068, filed Sep. 8, 2003, and published as WO 2004/023100,sets forth the finding that CD56 is an extracellular marker forprogenitors of pancreatic β cells, and describes in detail methods ofselecting CD56-positive cells. Without repeating the entirety of WO2004/023100, which is incorporated herein by reference, CD56-positivecells can be selected, for example, by use of CD56-specific antibodies,including antibodies that specifically binds an oligosaccharide linkedto the CD56 protein, as well as by use of lectins that specifically bindto such an oligosaccharide. Antibodies to CD56 are commerciallyavailable from a variety of sources, including Sigma-Aldrich (St. Louis,Mo.), Ancell Corp. (Bayport, Minn.), Diagnostic BioSystems (Pleasanton,Calif.), and Biocare Medical (Concord, Calif.). The selection forCD56-positive cells can be made before or after the selection ofEphA4-positive cells, as the practitioner finds convenient. Cells thatexpress both EphA4 and CD56 are expected to be particularly useful inthe methods and compositions of the present invention.

As can be appreciated from the disclosure provided above, the presentinvention has a wide variety of applications. Accordingly, the followingexamples are offered for illustration purposes and are not intended tobe construed as a limitation on the invention in any way.

EXAMPLES Example 1

This Example sets forth materials and methods used in studies underlyingthe present invention.

A. Organ Procurement

Pancreatic cells are isolated from cadaver pancreases. Organ harvestingis orchestrated by United Network for Organ Sharing (“UNOS”) and localorgan donor organizations. Only donors with signed consent forms forresearch are used.

For harvesting the pancreas, the abdominal aorta is cannulated below thejunction of the renal artery, and the portal perfusion is cannulated viathe inferior mesentery vein. The cannula is inserted into the portalvein (PV) to the level above the junction of the splenic vein (SV) tothe PV. A loose 2-0 tie is put around the SV at the junction with theportal vein, and another loose 2-0 tie is put around the splenic artery(SA). The SV tie is ligated and cut open on the spleen side immediatelybefore the perfusion starts. This makes the pancreas perfusion moreefficient without aortic/portal double end pressure which may damage theislets. It also allows all the portal perfusant to go into the liver andavoids draining the perfusant from the spleen and pancreas into theliver. The lesser sac is opened and a normal saline (“NS”) slush isapplied over the pancreas. After 1 L of aorta perfusion, the SA isligated. The pancreas should be well protected when the liver andkidneys are harvested. The pancreas is retrieved with the proceduresknown and used in the art for pancreas transplants.

The organ is stored in a plastic bag filled with UW solution (Universityof Wisconsin solution, known as “UW solution”, or “Belzer UW” is a coldstorage solution for organ preservation. See, e.g., Uhlmann et al., JSurg Res. 105(2):173-80 (2002), Southard et al., Transplantation.49(2):251-7 (1990), Fridell et al., Transplantation. 77(8):1304-1306(2004), Inui et al., Pancreas. 23(4):382-386 (2001), and Matsumoto etal., Transplant Proc. 36(4):1037-9 (2004). The formula is available onthe internet by entering “http://www.” followed by“surgery.wisc.edu/transplanthesearch/southard/UWFormula”. Belzer UWsolution is commercially available under the name ViaSpan® (BarrLaboratories, Inc., Pomona, N.Y.)) and set in a Nalgene® jar withsterile NS slush, or directly stored in a Nalgene® jar soaked in betweenO₂-saturated-perfluorocarbon (PFC) and UW solutions for transportation(placing pancreatic tissue between UW solution and a perfluorocarbon isknown as the two-layer system and is taught in, e.g., Matsumoto et al.,supra, Deai et al., Kobe J Med Sci 45:191-19-9 (1999), Hering et al.,Transplantation 74: 1813-1816 (2002), Ricordi et al., Transplantation75: 1524-1527 (2003), and Lakey et al., Transplantation 74: 1809-1811(2002). See generally, Shapiro, J Am Soc Nephrol 14:2214-2216 (2003)).

B. Pancreas Digestion

The islets are isolated by enzymatic pancreas digestion. One vial ofLiberase (0.5 g, Roche) is dissolved in 333 ml of HBSS (1.5 mg/ml, 37°C.) and infused into the pancreas via ductal cannulation(s). The organis incubated in an 800 ml tempering beaker at 37° C. for 10-20 minutesuntil the tissue becomes soft.

The semi-digested tissue mass is transferred into the metal digestionchamber and automatic circulating digestion started. Tissue isdissociated by agitation of the digestion chamber.

When the majority of islets have been released from the surroundingtissue, the digestant is collected and diluted with Medium A10 (10%fetal bovine serum in RPMI). The digestion procedure takes about 30minutes. The cells are washed with A10 three times at 4° C., 1,000 rpm,2 minutes, and then go through the cell separation procedure.

C. Pancreatic Cell Separation

The pellet resulting from the washing and centrifugation proceduredescribed in the preceding paragraph is mixed with 320 ml PancreaticIslet Purification Solution (“PIPS”) (a 13.7% solution of Nycodenz® AG(Axis-Shield PoC AS, Oslo, Norway; Nycodenz® is a centrifuge densitygradient solution with the systemic name5-(N-2,3-dihyroxypropylacetamide)-2,4,6-tri-iodo-N,N′-bis(2,3dihydroxypropyl)isophtalamide) prepared in ViaSpan® Belzer UW solution(density 1.114) and set on ice for 10 minutes.

Each of eight 250 ml flat-bottom centrifuge tubes are filled with 70 mlPIPS (density 1.090). Forty ml of cell/PIPS suspension is thenunder-laid into each tube. Sixty ml of RPMI 1640 with 2% FBS isover-laid on top of the PIPS. The tubes are centrifuged for six minuteswithout braking, using a Sorvall RC-3C Plus with a 05, ARC rotor at1,500 rpm.

The upper interface (A layer, purified islets), lower interface (Blayer, mixture of entrapped islets, fragmental islets, acinar and ductalcells) and the pellet (mainly acinar and ductal cells) are collectedseparately. The cells are washed two more times with Medium A10 and thenused as desired.

D. Antibody and Cocktail Assembly

EphA4 murine IgG monoclonal antibody is purchased from BD Biosciences(Catalog #s41820-050). Antibody cocktails are prepared according to theprotocol from StemCell Technologies, Vancouver, Canada. 15 μg of EphA4murine IgG 1 monoclonal antibody dissolved in 500 sterile phosphatebuffered saline (PBS) containing 2% FBS (fetal bovine serum) is added toa 1.5 mL polypropylene tube. 100 μL of component A (StemCellTechnologies) and 100 μL of component B (StemCell Technologies) aresequentially added to the vial. The vial is placed into a 37° C.incubator overnight. The vial is brought to a final volume of 1.0 ml byadding sterile PBS.

E. Cell Selection Procedure

This procedure is used for processing up to 2.5×10⁸ cells perseparation. Nucleated cell suspensions are prepared at a concentrationof 1×10⁸ nucleated cells/ml in PBS containing 2% FBS. Conveniently, thecells are separated using the EasySep™ system (StemCell Technologies,Inc., Vancouver, BC, Canada). For use in the EasySep™ system, the cellsare placed in 12×75 mm polystyrene tubes (Falcon® 5 mL PolystyreneRound-Bottom Tubes, Becton Dickinson, Catalogue #2058). The EasySep™system is an immunomagnetic cell selection procedure that uses specificantibodies and tiny FACS-compatible magnetic nanoparticles in acolumn-free magnetic system. The assembled positive selection cocktailis added to the cell suspension at 100 μl/ml cells. The cells are mixedwell and incubated at room temperature for 15 minutes. EasySep™ MagneticNanoparticles (StemCell Technologies) are mixed gently to ensure thatthey are in a uniform suspension by pipetting up and down more than 5times (vortexing is not recommended). The nanoparticles are added to thecells at the ratio of 50 μL/mL cells. The cells are mixed well andincubated at room temperature for 10 minutes. Cell suspension is broughtto a total volume of 2.5 mL by adding PBS containing 2% FBS. The cellsare mixed in the tube by gently pipetting up and down 3-4 times. Thecells are placed into the magnet and set aside for 5 minutes. In onecontinuous motion, the magnet and tube are inverted, and the supernatantfraction is poured off. The magnetically labeled cells remain inside thetube, held by the magnetic field of the EasySep™ magnet. The magnet andtube are left inverted for 2-3 seconds then returned to an uprightposition. The tube is removed from the magnet and 2.5 mL of recommendedmedium are added. The cell suspension is mixed by gently pipetting it upand down 2-3 times. The tube is placed back in the magnet and set asidefor five minutes. This procedure is repeated for a total of three5-minute separations in the magnet. The tube is removed from the magnetand the cells are resuspended in an appropriate amount of a chosenmedium. The positively selected cells are now ready for use.

Example 2

A. Eph Sorting Enhances Pancreatic Phenotype of Cultured PancreaticCells

1. Cell Culture and Cell Selection

The procedures of Eph sorting and cell culture are shown in FIG. 1.Human pancreatic HD469B (P0) cells were seeded in 10 cm plates inSM95/M7(4:1) medium and cultured for 6 days at 37° C. Medium was changedevery 2 days. On day 6, the HD469B cells were trypsinized and washedwith PBS. The cells were sorted with Eph antibody coated with MagneticNanoparticles (StemCell Technologies) prepared as described inExample 1. The sorted Eph positive cells (P1) (about 5% of original cellpopulation) and the Eph negative cells were cultured in SM95/M7 for 3days. At P2, The Eph sorted cells were cultured in SM95/M7(4:1) with 20μg/ml Wnt3a proteins for 5 days. On day 5, cells were trypsinized andwashed with PBS. Small portions of both Eph positive and negative cellswere harvested for RNA isolation for gene expression analysis byreal-time PCR. This procedure was performed at each cell passage fromP2-P5. The majority of Eph-sorted cells were passed into P3 and culturedfor three days in SM95/M7(4:1) with 20 μg/ml Wnt3a proteins. On passage4 and 5, each of the four group cells were divided into 3 plates withdifferent coating conditions: regular (control), fibronectin and extracellular matrix (ECM) coated plates. All cells were cultured inSM95/M7(9:1) for 3 day for each passage. At passage 6, cells werecultured in MM1 and MM2 medium for 3 days, respectively. After culturein MM2, cells were collected for isolation of total RNA.

2. Expression of EphA4 in Adult Pancreas and Primary Pancreatic CellCultures

EphA4 plays important role of sorting and cell specification duringdevelopment of adult tissues. To identify the expression of EphA4 inadult pancreas and cell culture, immunofluorenscence and ICC studies ofadult pancreas and primary pancreatic cells were performed. As shown inFIG. 2A, EphA4 positive cells were restricted to some of islet cells andno positive staining was found in acinar cells. Expression of EphA4 wasalso found in a subgroup of cultured pancreatic cells (FIG. 2C).

3. Comparison of Expression of Insulin in the EphA4+ and EphA4− Cells

Expression of insulin in EphA4 sorted cells was evaluated by real-timePCR. The mRNA levels of insulin in EphA4 positive cells were 9-112 timeshigher than the EphA4 negative cells (FIGS. 3 a and b). The highexpression of insulin maintained the EphA4 positive cells throughsequential (P2-P6) (FIG. 3, Table 1). Since EphA4-positive cells onlyrepresent about 5% of the original cell population, β-cell lineage cellswere greatly enriched by EphA4 sorting.

TABLE 1 Expression of Insulin in EphA4 Positive and Negative SortedCells at Different Cell Passages Insulin expression Insulin β-actinInsulin/β- ratio (EphA4+/ HD469B copy# Copy# actin ratio EphA4−) P2EphA4+ 6,046,000 25,190,000 2.400E−01 14 P2 EphA4− 184,500 11,030,0001.673E−02 1 P3 EphA4+ 425,800 6,108,000 6.971E−02 9 P3 EphA4− 26,1803,523,000 7.431E−03 1 P4 EphA4+ 569,000 14,390,000 3.954E−02 22 P4EphA4− 21,970 11,940,000 1.840E−03 1 P5 EphA4+ 16,200 373,400 4.339E−0246 P5 EphA4− 1,025 1,089,000 9.412E−04 1 P6 EphA4+ 93,840 204,8004.582E−01 112 P6 EphA4− 1,847 449,900 4.105E−03 1

4. Comparison of PDX-1 mRNA Expression in the EphA4+ and EphA4− Cells

We compared the expression of PDX-1 in both EphA4 positive- and EphA4negative-cells during different cell passages by real-time PCR. As shownin FIG. 4 and Table 2, EphA4 positive cells have 6-10 times higher mRNAlevel of PDX-1 than EphA4 negative cells either at just after sorting(P2) or during the following passages (P3-P6). PDX-1 is expressed athigh level at early passage (P2). The expression of PDX-1 was graduallydecreased as increased number of cell passages (P3-P5). After culturingin differentiation media, the expression of PDX-1 returned to high levelin EphA4 positive cells. These data show that EphA4 can enhancepancreatic phenotype of pancreatic cells.

TABLE 2 Expression of PDX-1 in EphA4 Positive- and Negative- SortedCells during Different Cell Passages PDX-1 expression ratio (EphA4+/HD469B β-actin PDX-1 EphA4−) P2 EphA4+ 25,190,000 1054 10 P2 EphA4−11,030,000 105 1 P3 EphA4+ 6,108,000 131 6 P3 EphA4− 3,523,000 22 1 P4EphA4+ 14,390,000 101 7 P4 EphA4− 11,940,000 14 1 P5 EphA4+ 373,400 1infinity P5 EphA4− 1,089,000 0 1 P6 EphA4+ 204,800 8 8 P6 EphA4− 449,9001 1

5. Comparison of Expression of Nkx2.2 and Pax6 in the EphA4+ and EphA4−Cells

Two pancreatic progenitor markers, Nkx2.2 and Pax6, were used toevaluate our pancreatic culture cells. Expression of Nkx2.2 and Pax6 inboth EphA4 positive and negative cells was quantified by real-time PCR.The expression of Nkx2.2 in EphA4 positive cells was 2.5˜16 times higherthan EphA4 negative cells at different cell passages (FIG. 5 Table 3).As expected, expression of Pax6 in the EphA4 positive and negativesorted cells has a very similar pattern to and tendency as theexpression of Nkx2.2. EphA4 positive cells have 8.5˜64 times higherexpression of Pax6 than EphA4 negative sorted cells during all thepassages (FIG. 6, Table 3). These results show that the EphA4-selectedcells can greatly enrich the progenitor cells of a pancreatic cellculture.

TABLE 3 Expression of NKx2.2 in EphA4 -Positive and -Negative SortedCells at Different Cell Passages Nkx2.2 expression Pax6 expressionNkx2.2/β- ratio (EphA4+/ Pax6/β- ratio (EphA4+/ HD469B β-actin Nkx2.2actin ratio EphA4−) Pax6 actin ratio EphA4−) P2 EphA4+ 25,190,000 10874.315E−05 11 10960 4.351E−04 11 P2 EphA4− 11,030,000 42 3.770E−06 1 4454.037E−05 1 P3 EphA4+ 6,108,000 164 2.687E−05 16 1180 1.932E−04 9 P3EphA4− 3,523,000 6 1.672E−06 1 80 2.262E−05 1 P4 EphA4+ 14,390,000 664.577E−06 5 1066 7.408E−05 13 P4 EphA4− 11,940,000 11 9.062E−07 1 665.562E−06 1 P5 EphA4+ 373,400 1 1.379E−06 2.5 9 2.464E−05 10 P5 EphA4−1,089,000 1 5.481E−07 1 3 2.583E−06 1 P6 EphA4+ 204,800 11 5.498E−05 7197 9.624E−04 64 P6 EphA4− 449,900 3 7.619E−06 1 7 1.528E−05 1

Example 3 CD56/EphA4 Double Selection Further Promotes PancreaticPhenotype of Cultured Pancreatic Cells

A. CD56 Sorting

1. Cell Culture and Cell Selection

The procedures of CD56 sorting and cell culture are shown in FIG. 7.Human pancreatic HD469B (P0) cells were seeded in 10 cm plates inSM95/M7(4:1) medium and culture for 6 days at 37° C. Medium was changedevery 2 days. On day 6, the HD469B cells were trypsinized and washedwith PBS. The cells were sorted with anti-CD56 antibody coated withMagnetic Nanoparticles (StemCell Technologies) prepared as described inExample 1. The sorted CD56-positive cells (P1) (about 5% of originalcell population) and the CD56-negative cells were cultured in SM95/M7for 3 days. At P2, the CD56 sorted cells were cultured in SM95/M7(4:1)with 20 μg/ml Wnt3a proteins for 5 days. On day 5, cells weretrypsinized and washed with PBS. Small portions of the cells from bothCD56 positive and negative cells were harvested for RNA isolation forgene expression analysis by real-time PCR. This procedure was performedat each cell passage from P2-P5. The majority of CD56 sorted cells werepassed into P3 and cultured for three days in SM95/M7(4:1) with 20 μg/mlWnt3a proteins. On passage 4 and 5, each of the four group cells wasdivided into 3 plates with different coating conditions: regular(control), fibronectin and extra cellular matrix (ECM) coated plates.All cells were cultured in SM95/M7(9:1) for 3 day for each passage. Atpassage 6, cells were culture in MM1 and MM2 medium for 3 days,respectively. After culture in MM2, cells were collected for isolationof total RNA.

2. Comparison of Insulin Expression in the CD56+ and CD56− Cells

Expression of insulin in CD56 sorted cells was evaluated by real-timePCR. The mRNA levels of insulin in CD56 positive cells were 4˜12 timeshigher than in CD56 negative cells (FIG. 8, Table 4). The highexpression of insulin maintained the CD56 positive cells throughsequential (P2-P6) (FIG. 13). Since CD56 positive cells represent onlyabout 5% of the original cell population, β-cell lineage cells weregreatly enriched by CD56 sorting.

TABLE 4 Expression of Insulin in CD56 Positive and Negative Cells atDifferent Cell Passages Insulin expression Ins/β- ratio (EphA4+/ HD469BInsulin β-actin actin ratio EphA4−) P2 CD56+ 550,200 2,162,000 2.545E−0111 P2 CD56− 256,100 11,050,000 2.318E−02 1 P3 CD56+ 494,500 15,610,0003.168E−02 11 P3 CD56− 14,070 4,916,000 2.862E−03 1 P4 CD56+ 232,40013,360,000 1.740E−02 4 P4 CD56− 51,500 13,200,000 3.902E−03 1 P5 CD56+13,750 1,176,000 1.169E−02 10 P5 CD56− 1,608 1,408,000 1.142E−03 1 P6CD56+ 37,080 239,800 1.546E−01 12 P6 CD56− 4,849 380,200 1.275E−02 1

3. Comparison of PDX-1 Expression in the CD56+ and CD56− Cells

We compared the expression of PDX-1 in both CD56 positive and CD56negative cells during the cell different cell passages by real-time PCR.As shown in FIG. 9 and Table 5, CD56 positive cells have significantlyhigher mRNA level of PDX-1 than CD56 negative cells especially duringthe later passages (P3-P6). The PDX-1 expressions in the CD+ and CD−cells were at same level at P2 but PDX-1 expression in the CD+cells weregradually elevated as increased number of cell passages (P3-P5) andreached 26 times higher then CD− cells at P6 (Table 5). After culturingin differential medium, the expression of PDX-1 returned to high levelin CD56 positive cells.

TABLE 5 Expression of PDX-1 in CD56 -Positive and -Negative Sorted Cellsduring Different Cell Passages PDX-1 expression PDX/β- ratio (EphA4+/HD469B β-actin PDX-1 actin ratio EphA4−) P2 CD56+ 2,162,000 11 4.977E−061 P2 CD56− 11,050,000 64 5.750E−06 1 P3 CD56+ 15,610,000 143 9.167E−06 2P3 CD56− 4,916,000 20 3.975E−06 1 P4 CD56+ 13,360,000 49 3.641E−06 3 P4CD56− 13,200,000 15 1.123E−06 1 P5 CD56+ 1,176,000 3 2.173E−06 infinityP5 CD56− 1,408,000 0 0.000E+00 1 P6 CD56+ 239,800 6 2.604E−05 26 P6CD56− 380,200 0 1.021E−06 1

4. Comparison of Nkx2.2 and Pax6 Expression in the CD56+ and CD− Cells

Two pancreatic progenitor markers, Nkx2.2 and Pax6, were used toevaluate CD56 sorted cell cultures. Expression of Nkx2.2 and Pax6 inboth CD56 positive and negative cells were quantified by real-time PCR.The expression of Nkx2.2 in CD56 positive cells was 1.6˜7 times higherthan CD56 negative cells at different cell passages (FIG. 10, Table 6).Interestingly, expression of Pax6 in the CD56 positive and negativesorted cells has a very similar pattern and tendency as does theexpression of Nkx2.2. CD56 positive cells showed 4˜7 times higher Pax6expression than CD56 negative cells during the passages (FIG. 11, Table6). Thus, selection for CD56 positive cells can greatly enrich theprogenitor cells of a pancreatic cell culture.

TABLE 6 Expression of Nkx2.2 and Pax6 in CD56 Positive and NegativeSorted Cells during Different Cell Passages Nkx2.2 expression Pax6expression Nkx2.2/β- ratio (EphA4+/ Pax6/β- ratio (EphA4+/ HD469Bβ-actin Nkx2.2 actin ratio EphA4−) Pax6 actin ratio EphA4−) P2 CD56+2,162,000 10 4.820E−06 1.5 808 3.735E−04 7 P2 CD56− 11,050,000 363.271E−06 1 608 5.502E−05 1 P3 CD56+ 15,610,000 133 8.533E−06 7 15811.013E−04 7 P3 CD56− 4,916,000 6 1.162E−06 1 73 1.491E−05 1 P4 CD56+13,360,000 36 2.703E−06 1.6 544 4.070E−05 4 P4 CD56− 13,200,000 221.644E−06 1 121 9.144E−06 1 P5 CD56+ 1,176,000 2 1.481E−06 infinity 282.371E−05 6 P5 CD56− 1,408,000 0 0.000E+00 1 6 3.960E−06 1 P6 CD56+239,800 5 1.958E−05 6.5 75 3.146E−04 4 P6 CD56− 380,200 1 3.019E−06 1 287.412E−05 1B. Secondary Sorting with EPHA4 Antibody

1. Cell Culture and Cell Selection

The scheme of cell culture and cell sorting is shown in FIG. 12. HD469B(P0) cells were seeded in 10 cm plates in SM95/M7(4:1) medium andculture for 6 days at 37° C. Medium was changed every 2 days. On day 6,the HD469B cells were trypsinized and washed with PBS. The cells sortedwith anti-CD56 antibody were coated with Magnetic Nanoparticles(StemCell Technologies) as described in Example 1. The sorted CD56positive cells (P1) (about 10% of original cell population) and the CD56negative cells were cultured in SM95/M7 for 3 days. The cells weretrypsinized and washed with PBS. A small portion of the cells (10% ofcell population) was passed into a 10 cm plate (CD56 positive P2) inSM95/M7(4:1) with 20 μg/ml Wnt3a proteins (R&D Systems, Inc). Themajority of the cells were sorted with an antibody to EphA4 (BDBiosciences) coated with Magnetic Nanoparticles (StemCell Technologies)prepared as described in Example 1. The sorted CD56-EphA4 positive cells(P2) and CD56-EphA4 negative cells (P2) were cultured in SM95/M7(4:1)with 20 μg/ml Wnt3a proteins for 5 days. On day 5, CD56/EphA4 cells(P2), CD56/EphA4 negative cells (P2), CD56 positive cells (P2) and CD56negative cells (P2) were trypsinized and washed with PBS. Small portionsof cells from each group were harvested for RNA isolation for geneexpression analysis by real-time PCR. This procedure was performed ateach cell passage from P2-P5. The majority of the cells in all fourgroups were passed into P3 and cultured for three days in SM95/M7(4:1)with 20 μg/ml Wnt3a proteins. On passage 4 and 5, each of the four groupcells were divided into 3 plates with different coating conditions,regular (control), fibronectin, and extra cellular matrix (ECM) coatedplates. All cells were cultured in SM95/M7(9:1) for 3 day for eachpassage. At passage 6, cells were culture in MM1 and MM2 medium for 3days, respectively. After culture in MM2, cells were collected forisolation of total RNA.

2. Insulin Expression in the CD56/EphA4 Double Selected Cells

To characterize the CD56/EphA4 double positive cells, we first measuredmRNA level of insulin among different cell groups by real time PCR. Asshown in FIG. 13 and Table 7, CD56-EphA4 double positive cells have thehighest expression of insulin among all the cell groups after sorting atP1 (FIG. 13A). The expression of insulin in CD56+/EphA4+ cells (0.46)were around two-fold higher compared to either CD56 single positive(0.25) or EphA4 single positive (0.24) cells (FIG. 13A). Significantly,the double positive cells maintained high expression of insulin throughsequential passages with different culture conditions (FIG. 13B, C, D,Table 7). The mRNA levels of insulin in CD56+/EphA4+ cells were always2˜5 times higher than either CD56 or EphA4 single positive sorted cellsduring the passages. These results show that CD56/EphA4 double selectioncan enhance pancreatic endocrine phenotype of both CD56 positive orEphA4 positive cells.

Recently, another group used a method for genetic lineage tracing todetermine the contribution of stem cells to β-cells. They showed theinsulin producing cells are the major source of new β-cells during adultlife in mice. These insulin producing β-cells progenitors are derivedfrom either pre-exiting β-cells which were transiently dedifferentiatedor stem cells which produce insulin.

TABLE 7 Insulin Expression of CD56, EphA4, and CD56/EphA4 double sortedcells at different passages Ins/β- HD469B Insulin β-actin actin ratio P2CD56+ 550,200 2,162,000 2.545E−01 P2 CD56− 256,100 11,050,000 2.318E−02P2 EphA4+ 6,046,000 25,190,000 2.400E−01 P2 EphA4− 184,500 11,030,0001.673E−02 P2 CD56+ EphA4+ 9,960,000 21,850,000 4.558E−01 P2 CD56+ EphA4−98,210 2,095,000 4.688E−02 P3 CD56+ 494,500 15,610,000 3.168E−02 P3CD56− 14,070 4,916,000 2.862E−03 P3 EphA4+ 425,800 6,108,000 6.971E−02P3 EphA4− 26,180 3,523,000 7.431E−03 P3 CD56+ EphA4+ 1,878,00011,900,000 1.578E−01 P3 CD56+ EphA4− 51,100 7,943,000 6.433E−03 P4 CD56+232,400 13,360,000 1.740E−02 P4 CD56+ ECM 148,700 7,160,000 2.077E−02 P4CD56+ Fib 216,400 13,280,000 1.630E−02 P4 CD56− 51,500 13,200,0003.902E−03 P4 CD56− ECM 8,556 7,054,000 1.213E−03 P4 CD56− Fib 29,93012,000,000 2.494E−03 P4 EphA4+ 569,000 14,390,000 3.954E−02 P4 EphA4+ECM 318,900 13,320,000 2.394E−02 P4 EphA4+ Fib 236,700 4,701,0005.035E−02 P4 EphA4− 21,970 11,940,000 1.840E−03 P4 EphA4− ECM 8,33914,950,000 5.578E−04 P4 EphA4− Fib 9,268 9,661,000 9.593E−04 P4 CD56+EphA4+ 1,203,000 17,010,000 7.072E−02 P4 CD56+ EphA4+ ECM 812,60011,990,000 6.777E−02 P4 CD56+ EphA4+ Fib 910,600 12,530,000 7.267E−02 P4CD56+ EphA4− 22,750 13,010,000 1.749E−03 P4 CD56+ EphA4− ECM 10,8809,459,000 1.150E−03 P4 CD56+ EphA4− Fib 6,673 5,468,000 1.220E−03 P6CD56+ 37,080 239,800 1.546E−01 P6 CD56+ ECM 28,150 147,800 1.905E−01 P6CD56+ Fib 72,330 1,022,000 7.077E−02 P6 CD56− 4,849 380,200 1.275E−02 P6CD56− ECM 5,939 450,300 1.319E−02 P6 CD56− Fib 3,899 337,200 1.156E−02P6 EphA4+ 93,840 204,800 4.582E−01 P6 EphA4+ ECM 62,210 135,0004.608E−01 P6 EphA4+ Fib 41,600 85,440 4.869E−01 P6 EphA4− 1,847 449,9004.105E−03 P6 EphA4− ECM 541 255,400 2.117E−03 P6 EphA4− Fib 2,076367,100 5.655E−03 P6 CD56+ EphA4+ 395,500 1,016,000 3.893E−01 P6 CD56+EphA4+ ECM 172,500 231,000 7.468E−01 P6 CD56+ EphA4+ Fib 101,800 114,1008.922E−01 P6 CD56+ EphA4− 885 101,100 8.753E−03 P6 CD56+ EphA4− ECM2,323 382,700 6.070E−03 P6 CD56+ EphA4− Fib 2,351 375,500 6.261E−03

3. Expression of PDX-1 in the CD56/EphA4 Double Selected Cells

We analyzed the expression of pancreatic gene PDX-1 among different cellgroups during the cell passages. PDX-1 plays important role duringpancreatic development lineage including the specification,proliferation and differentiation of pancreatic cell types. It activatesthe transcription of many β-cell genes involved in glucose homeostasisincluding insulin, glucokinase and the glucose transporter GLUT2. PDX-1is expressed during pancreatic development with high levels ofexpression in both early embryonic endodermally derived cells anddifferentiated β-cells. A conditional PDX-1 null mouse modeldemonstrated that PDX-1 is not only required for proper specification ofdifferent pancreatic cell types, but also for pancreatic progenitorcells to differentiate into β-cells (Holland et al., Proc Natl Acad Sci99(19):12236-41 (2002)). mRNA levels of PDX-1 were measured amongdifferent cell culture groups by real-time PCR. As shown in FIG. 14 andTable 8, CD56/EphA4 double positive cells showed higher PDX-1 expressionthan CD56 positive cells through out all the passages (P2-P6). Theexpression of PDX-1 in CD56/EphA4 double positive cells was highercompared with the EphA4 single positively sorted cells during P3˜P6. AtP2, both CD56+/EphA4+ and EphA4+ cells had similar levels of PDX-1expression. These data further support that EphA4 can further enhancepancreatic endocrine phenotype of CD56 selected cells.

TABLE 8 Expression of PDX-1 in CD56−, EphA4−, and CD56/EphA4 double-sorted cells at different passages PDX/β- HD469B PDX-1 β-actin actinratio P2 CD56+ 11 2,162,000 4.97E−06 P2 CD56− 64 11,050,000 5.75E−06 P2EphA4+ 1054 25,190,000 4.18E−05 P2 EphA4− 105 11,030,000 9.49E−06 P2CD56+ EphA4+ 738 21,850,000 3.37E−05 P2 CD56+ EphA4− 6 2,095,0002.78E−06 P3 CD56+ 143 15,610,000 9.16E−06 P3 CD56− 20 4,916,000 3.97E−06P3 EphA4+ 131 6,108,000 2.15E−05 P3 EphA4− 22 3,523,000 6.33E−06 P3CD56+ EphA4+ 457 11,900,000 3.84E−05 P3 CD56+ EphA4− 39 7,943,0004.87E−06 P4 CD56+ 49 13,360,000 3.641E−06 P4 CD56+ ECM 20 7,160,0002.778E−06 P4 CD56+ Fib 42 13,280,000 3.127E−06 P4 CD56− 15 13,200,0001.123E−06 P4 CD56− ECM 6 7,054,000 7.851E−07 P4 CD56− Fib 5 12,000,0004.363E−07 P4 EphA4+ 101 14,390,000 6.984E−06 P4 EphA4+ ECM 10713,320,000 8.048E−06 P4 EphA4+ Fib 25 4,701,000 5.269E−06 P4 EphA4− 1411,940,000 1.157E−06 P4 EphA4− ECM 6 14,950,000 4.131E−07 P4 EphA4− Fib5 9,661,000 5.612E−07 P4 CD56+ EphA4+ 249 17,010,000 1.461E−05 P4 CD56+EphA4+ ECM 173 11,990,000 1.443E−05 P4 CD56+ EphA4+ Fib 170 12,530,0001.355E−05 P4 CD56+ EphA4− 10 13,010,000 7.636E−07 P4 CD56+ EphA4− ECM 79,459,000 7.742E−07 P4 CD56+ EphA4− Fib 3 5,468,000 4.702E−07 P6 CD56+ 6239,800 2.604E−05 P6 CD56+ ECM 3 147,800 2.179E−05 P6 CD56+ Fib 361,022,000 3.494E−05 P6 CD56− 0 380,200 1.021E−06 P6 CD56− ECM 1 450,3001.528E−06 P6 CD56− Fib 0 337,200 0.000E+00 P6 EphA4+ 8 204,800 3.695E−05P6 EphA4+ ECM 8 135,000 5.785E−05 P6 EphA4+ Fib 0 85,440 3.538E−06 P6EphA4− 1 449,900 2.071E−06 P6 EphA4− ECM 2 255,400 6.143E−06 P6 EphA4−Fib 0 367,100 0.000E+00 P6 CD56+ EphA4+ 123 1,016,000 1.207E−04 P6 CD56+EphA4+ ECM 14 231,000 6.039E−05 P6 CD56+ EphA4+ Fib 3 114,100 2.430E−05P6 CD56+ EphA4− 0 101,100 9.066E−07 P6 CD56+ EphA4− ECM 1 382,7001.662E−06 P6 CD56+ EphA4− Fib 0 375,500 0.000E+00

4. Expression of Nkx2.2 and Pax6 in the CD56/EphA4 Double Selected Cells

Two pancreatic progenitor markers, Nkx2.2 and Pax6, were also used toevaluate our pancreatic culture cells. Nkx2.2 is a homeodomaintranscription factor expressed in early stage of pancreaticdevelopments. It is expressed in the pancreatic bud until E13 when itbecomes localized to the neurogenin3-expressing progenitor cells. Nkx2.2null mice have a complete absence of insulin-producing cells (Sussel etal, Development, 125(12):2213-21 (1998)). Pax6 is a late factor afterneurogenin3 expression and in conjunction with hormone gene expression.Pax6 is critical to the development and maintenance of the finaldifferentiated islet cell phenotypes. Loss of Pax6 causes defects in thegeneration of all endocrine cell types (Ahlgren et al., Nature385:257-60 (1997)). Expression of Nkx2.2 and Pax6 in different cellgroups was quantified by real-time PCR. The expression of Nkx2.2 inCD56/EphA4 positive cells was higher than either CD56 or EphA4 positivecell at different cell passages (FIG. 15). As expected, the expressionof Pax6 in the different sorted cells had a very similar pattern to andtendency as the expression of Nkx2.2. CD56/EphA4 positive cells havehigher expression of Pax6 than either CD56 or EphA4 sorted cells duringall the passages (FIG. 16). These results show that the EphA4 selectioncells can further enrich the progenitor cells of either CD56 positive orEphA4 positive cells.

TABLE 9 Expression of NKx2.2 and Pax6 in CD56, EphA4, or Double SortedCells at Different Cell Passages Nkx2.2/β- Pax6/β- HD469B β-actin Nkx2.2actin ratio Pax6 actin ratio P2 CD56+ 2,162,000 10 4.820E−06 8083.735E−04 P2 CD56− 11,050,000 36 3.271E−06 608 5.502E−05 P2 EphA4+25,190,000 1087 4.315E−05 10960 4.351E−04 P2 EphA4− 11,030,000 423.770E−06 445 4.037E−05 P2 CD56+ EphA4+ 21,850,000 1092 4.998E−05 223601.023E−03 P2 CD56− EphA4− 2,095,000 2 1.151E−06 130 6.224E−05 P3 CD56+15,610,000 133 8.533E−06 1581 1.013E−04 P3 CD56− 4,916,000 6 1.162E−0673 1.491E−05 P3 EphA4+ 6,108,000 164 2.687E−05 1180 1.932E−04 P3 EphA4−3,523,000 6 1.672E−06 80 2.262E−05 P3 CD56+ EphA4+ 11,900,000 4824.051E−05 4714 3.961E−04 P3 CD56− EphA4− 7,943,000 8 1.008E−06 1481.866E−05 P4 CD56+ 13,360,000 36 2.703E−06 544 4.070E−05 P4 CD56−13,200,000 22 1.644E−06 121 9.144E−06 P4 EphA4+ 14,390,000 66 4.577E−061066 7.408E−05 P4 EphA4− 11,940,000 11 9.062E−07 66 5.562E−06 P4 CD56+EphA4+ 17,010,000 159 9.371E−06 2607 1.533E−04 P4 CD56− EphA4−13,010,000 6 4.396E−07 53 4.071E−06 P5 CD56+ 1,176,000 2 1.481E−06 282.371E−05 P5 CD56− 1,408,000 0 0.000E+00 6 3.960E−06 P5 EphA4+ 373,400 11.379E−06 9 2.464E−05 P5 EphA4− 1,089,000 1 5.481E−07 3 2.583E−06 P5CD56+ EphA4+ 4,701,000 35 7.375E−06 321 6.826E−05 P5 CD56+ EphA4−10,930,000 1 5.500E−08 10 9.552E−07 P6 CD56+ 239,800 5 1.958E−05 753.146E−04 P6 CD56− 380,200 1 3.019E−06 28 7.412E−05 P6 EphA4+ 204,800 115.498E−05 197 9.624E−04 P6 EphA4− 449,900 3 7.619E−06 7 1.528E−05 P6CD56+ EphA4+ 1,016,000 99 9.727E−05 1378 1.356E−03 P6 CD56+ EphA4−101,100 0 2.419E−06 5 4.539E−05

Example 4 In Vitro and In Vivo Characterizations of CD56 or EphA4 SingleSorted and CD56/EphA4 or EphA4/CD56 Double Sorted Pancreatic Cells

A. Materials and Methods

Pre-purified cells from digested human pancreas donor HD496 werecultured with SM95+M7 (4:1) at P0. When the culture dish reachedconfluence, the cells were divided into two portions, one portion wassorted with CD56 the other one sorted with EphA4. Both positively andnegatively sorted cells were collected and put in P1 culture. When theculture dish reached confluence, cells were collected and the CD56− andEphA4− cells and half of the CD56+ and EphA4+ cells were directlypassaged to P2, and the other half of the positively sorted cells wentthrough secondary sorting with different antibodies. Finally thesortings yielded eight cell groups (FIG. 17).

-   CD56+-   CD56−-   EphA4+-   EphA4−-   CD56+/EphA4+-   CD56+/EphA4−-   EphA4+/CD56+-   EphA4+/CD56−

B. Part 1. In Vitro Characterization of CD56 or EphA4 Single Sorted andCD56/EphA4 or EphA4/CD56 Double Sorted Pancreatic Cells

Cells were divided into groups (FIG. 17). Group1 was first sorted withCD56 at the end of P0, and then sorted with EphA4 at the end of P1. Theother group was first sorted with EphA4 at the end of P0 and then sortedwith CD56 at the end of P1. The cells were then divided into eightgroups, as follows:

-   CD56+-   CD56−-   EphA4+-   EphA4−-   CD56+/EphA4+-   CD56+/EphA4−-   EphA4+/CD56+-   EphA4+/CD56−

(1). In Vitro Characterizations

The different cell group samples were collected for qRT-PCR to test geneexpressions of CD56, EphA4, Insulin, Glucagon, PDX1, Glut2, CK19 andAmylase.

The qRT-PCR samples were collected at four different time points, namelyP0 before sorting, P0 immediately after sorting, P1 before 2^(nd)sorting and P1 after 2^(nd) sorting.

(2). Results

After first sorting, the insulin expressions of CD56 positive and EphA4positive cells were 22 and 15 times higher than the negative onesrespectively, while the glucagon expressions were 27 and 17 times higherin the positive cells. The GluT2 expression in the CD56 positive and Ephpositive cells was 3-4 times higher than the negative ones. After secondsorting, the insulin and glucagon gene expressions of CD56/EphA4 doublepositive cells reached or exceeded the P0 level and were 4˜100 timeshigher than the other groups (Table 10).

TABLE 10 Gene Expression of CD56, EphA4, Insulin, Glucagon, PDX1, Glut2,CK19, and Amylase in CD56, EphA4, or Double Sorted Cells at DifferentCell Passages Ins/β- Gcg/β- PDX/β- GLUT2/β- Amy/β- NCAM/β- CK19/β-Pax4/β- SA22/β- actin actin actin actin actin actin actin actin actinCell ratio ratio ratio ratio ratio ratio ratio ratio ratio P0, beforeHD495B 6.7E−01 1.2E−02 3.3E−03 2.5E−04 6.5E−02 6.6E−03 1.9E−01 0.0E+004.2E−04 sorting P0, post- CD56+ 4.0E+00 5.2E−02 1.0E−02 8.5E−04 1.1E−012.8E−02 3.7E−01 0.0E+00 8.0E−04 1st sorting CD56− 1.8E−01 1.9E−034.1E−03 2.7E−04 9.4E−02 1.9E−03 1.9E−01 0.0E+00 2.1E−04 EphA4+ 4.5E+005.7E−02 1.4E−02 8.0E−04 1.0E−01 3.0E−02 3.5E−01 3.5E−06 9.1E−04 EphA4−3.0E−01 3.3E−03 4.4E−03 2.2E−04 1.0E−01 1.8E−03 1.7E−01 0.0E+00 2.8E−04P1, before CD56+ 6.1E−01 1.7E−02 1.5E−03 1.5E−04 1.5E−02 3.6E−03 2.9E−015.2E−06 2.2E−03 2nd sorting CD56− 2.7E−02 8.6E−04 6.8E−04 1.3E−041.3E−02 9.4E−04 2.2E−01 0.0E+00 8.9E−04 EphA4+ 1.0E+00 2.9E−02 1.4E−031.7E−04 1.3E−02 5.2E−03 2.6E−01 1.6E−05 2.2E−03 EphA4− 2.3E−02 9.4E−047.6E−04 8.4E−05 1.8E−02 1.4E−03 2.6E−01 0.0E+00 1.1E−03 P1, post-CD56+/Eph+ 6.4E−01 3.1E−02 1.5E−03 2.6E−04 1.1E−02 7.2E−03 4.2E−014.8E−06 3.6E−03 2nd-sorting CD56+/Eph− 1.6E−01 5.3E−03 9.7E−04 1.7E−041.6E−02 2.5E−03 4.5E−01 0.0E+00 1.5E−03 CD56−/Eph+ 1.5E−01 8.7E−032.5E−03 3.9E−04 1.9E−02 7.8E−03 4.0E−01 0.0E+00 6.5E−03 CD56−/Eph−4.7E−03 2.5E−04 7.7E−04 1.1E−04 1.5E−02 9.7E−04 3.6E−01 0.0E+00 7.8E−04Eph+/CD56+ 1.1E+00 3.2E−02 1.2E−03 1.2E−04 1.4E−02 5.9E−03 3.4E−010.0E+00 3.5E−03 Eph+/CD56− 2.6E−01 1.2E−02 6.6E−04 1.1E−04 1.3E−022.5E−03 3.3E−01 0.0E+00 1.5E−03 Eph−/CD56+ 3.5E−02 4.3E−03 2.1E−033.2E−04 1.4E−02 2.9E−03 3.9E−01 0.0E+00 2.7E−03 Eph−/CD56− 2.6E−021.4E−03 5.0E−04 5.8E−05 1.3E−02 9.2E−04 2.7E−01 0.0E+00 1.1E−03

(3). Summary:

-   -   Both EphA4+ and CD56+ cells have much higher        insulin/glucagon/PDX-1/GLUT2 levels, suggesting that the        CD56/EphA4 sorting can enrich endocrine lineage cells, which        include both β and α cells.    -   Overlapping between CD56 and EphA4 expression suggests that        these markers co-exist on the surface of certain cell        population.    -   CD56 and EphA4 sorting did not significantly affect Amylase/CK19        expressing cells, suggesting that they are exclusive markers for        endocrine cells.    -   EphA4 levels increased after proliferation in GM1SB, which        suggests that EphA4+ cells might represent some expandable        endocrine cell population.        B. Part 2. Comparisons of Insulin Expression in CD56/EphA4        Single Sorted and CD56/EphA4 or EphA4/CD56 Double Sorted        Pancreatic Cells

(1). Cells

Cell sorting procedure was as described above. All the cells werecultured in SM95+M7 (4:1, day P0) or SM95 in the following passages.

Cells were divided into two groups one group was sorted with CD56 at theend of P0 and then sorted with EphA4 at the end of P1. The other groupwas sorted with EphA4 at the end of P0 and then CD56 at P1.

Insulin qRT-PCR was tested for both groups at P0, P1 and P2 to comparewhether the different sorting sequence would change the outcome.

(2). Results

As shown in FIG. 26, both groups had similar patterns of insulinexpression. The different sorting sequence did not change the outcome.

B. Part 3 Comparisons of Insulin Expression in SM95 and DM CulturedCD56/EphA4 Single Sorted and CD56/EphA4 or EphA4/CD56 Double SortedPancreatic Cells

(1). Cells

Cell sorting procedure was as described above. All the cells werecultured in SM95+M7 (4:1, ay P0) or SM95 in the following passages.

Cells were divided into two groups; one group was sorted with CD56 atthe end of P0 and then sorted with EphA4 at the end of P1. The othergroup was sorted with EphA4 at the end of P0 and then with CD56 at P1.The cells were cultured in SM95 before sorting. After the secondsorting, the sorted cells were divided into two groups. One group waskept in SM95 and the other group was treated with DM (differentiationmedium) for three days. The formula of DM is as followsBasal medium: DMEM/F12(1:1)+N2+B27

-   -   (note: DMEM mixed with Ham's F12 media in a 1:1 ratio is        available premixed from a number of suppliers, including        AthenaES, Baltimore, Md., HyClone, Logan, Utah, and Mediatech,        Inc., Herndon, Va. N2 and B27 are commercially available media        additives.)        Supplements:

-   10 mM Nicotinamide (available from, e.g., Sigma-Aldrich, Inc., St.    Louis, Mo.);

-   10 ng/ml recombinant human growth hormone (available from, e.g.,    Humatrope®, Eli Lilly & Co., Indianapolis, Ind.);

-   200 nM of the peptide IGLHDPSHGTLPNGS;

-   10 ng/ml Exendin-4 (Epoch Biolabs, Sugar Land, Tex.);

-   2 ng/ml human recombinant betacellulin (R&D Systems, Inc.,    Minneapolis, Minn.); and,

-   100 nM Z-VAD-FMK (a pan-caspase inhibitor; Epoch Biolabs, Sugar    Land, Tex.)

Insulin qRT-PCR was tested for both groups at P2 to compare whether thedifferent culture media would change the outcome.

(2). Results

The insulin expression after DM treatment was twice as high as thenon-DM-treated groups and reached levels higher than that seen at P0(FIG. 27).

B. Part 4. In Vivo Characterization of EPHA4 Sorted and ProliferatedHuman Pancreatic Cells

(1). Cells

Cell sorting procedure was as described above. Human pancreatic cellswere seeded in SM95+M7 (4:1) for P0 culture. EphA4 sorting was performedat P1, and then the cells were proliferated in SM95 until P5. When theculture dish was confluent, cells were trypsinized and collected forencapsulation. After the cells were well mixed with Alginate solution,the mixture was dropped into calcium solution to form gel capsulesthrough an airflow head-jet. The capsules were then cultured in medium 4until transplant.

(2) Experimental Animals

Normal C57/black mice were made diabetic by a single intraperitonealinjection of streptozotocin at 220 mg/dl. When the blood glucose ofinjected animals reached 400 mg/dl or above, the encapsulated humanpancreatic cells were implanted into the peritoneal cavity through amid-line abdominal incision.

The transplanted animals were followed up by measuring blood glucoseweekly. The animals were sacrificed when their blood glucose reachednormal range (<200 mg/dl). Biopsy of pancreases and blood samples werecollected. Blood human C-peptide was measured for verifying the insulinsecretion of the grafts.

In one exemplar study, three animals were studied. One receivedpassage5-EphA4 positively sorted cells as described above, one was anon-transplanted diabetic mouse and the other one was normal control.

(3) Results

Table 11 and FIG. 28 present information about the mice in this study.The blood glucose of the transplanted animal showed a recovery frompre-Tx hyperglycemia (>400 mg/dl) to the near normal range (<300 mg/dl)within five weeks, while the non-transplanted diabetic mouse washyperglycemic through out the study period.

TABLE 11 Comparison of blood glucose (mg/dl) of diabetic C57 mousetransplanted with encapsulated P5 EphA4 sorted B cells (sorted at P0)and non-transplanted diabetic mouse Pre- Post-Tx Tx week 1 week 2 week 3week 4 Week 12 Tx mouse 454 295 382 378 370 280 Non-Tx 441 457 486 495501 died mouse

Table 12 shows the human blood C-peptide of the experimental animals.The blood human C-peptide test kit (ELISA) does not cross react withrodent C-peptide, so normally the rodent blood human-peptide should beless than 0.02, which is the test background. If the transplanted animalhas human C-peptide in blood, it means the human pancreatic cell graftis functioning. In this study, the EphA4+ cell mouse recipient had 0.64ng/ml human C-peptide in the blood, while the non-transplanted normalcontrol mouse only had some trace value (0.005 ng/ml) (Table 12).

TABLE 12 Human C-peptide concentrations in the blood of a transplantedmouse and a control normal mouse Blood Human C- Animal peptide (ng/ml)Recipient of EphA4+ Human 0.64 Pancreatic cells Normal(non-transplanted) control 0.005

(4) Conclusions

EphA4 sorting enriches all the pancreatic endocrine lineage cellsincluding both α- and β-lineage cells. This means these sorted cellshave the potential not only to cure hyperglycemia but also to preventhypoglycemia, which is a dangerous complication of diabetes. EphA4 andCD56 double sorting strengthened the endocrine cell enrichment effect.CD56 and EphA4 sorting methods make it possible to utilize the endocrinelineage cells from the human pancreas to selectively expand theendocrine cell population and develop regenerated islets.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A cell culture of propagating pancreatic cells produced by the stepsof: (a) isolating pancreatic cells from a pancreas, (b) a EphA4 cellselection, and (c) a CD56 cell selection, wherein the EphA4 cellselection comprises (1) contacting the pancreatic cells with an EphA4binding reagent; (2) selecting pancreatic cells that specifically bindto the EphA4 binding reagent; and (3) separating the selected pancreaticcells from pancreatic cells that do not bind the EphA4 binding reagentto obtain a culture of propagating EphAr pancreatic cells, and whereinthe CD56 cell selection comprises (x) contacting the pancreatic cellswith a CD56 binding reagent; (y) selecting pancreatic cells thatspecifically bind to the CD56 binding reagent; and (z) separating theselected pancreatic cells from pancreatic cells that do not bind theCD56 binding reagent to obtain a culture of propagating CD56⁺ pancreaticcells, wherein the propagating pancreatic cells have a capacity todifferentiate into insulin producing cells and wherein the CD56 cellselection is performed before or after contacting the pancreatic cellswith the EphA4 binding reagent.
 2. The cell culture of claim 1, whereinthe EphA4 binding reagent is labeled.
 3. The cell culture of claim 1,wherein the step of selecting is done by fluorescence activated cellsorting.
 4. The cell culture of claim 1, wherein the EphA4 cellselecting is done by panning.
 5. The cell culture of claim 1, whereinthe EphA4 binding reagent is an antibody that specifically binds to theEphA4 protein.
 6. The cell culture of claim 1, wherein the pancreas isfrom a human.
 7. A cell culture of claim 1, wherein the CD56 cellselection is performed before contacting the pancreatic cells with theEphA4 binding reagent.
 8. A cell culture of claim 1, wherein the CD56cell selection is performed after cells binding the EphA4 bindingreagent are separated from pancreatic cells that do not bind the EphA4binding reagent.
 9. A cell culture comprising a substantially homogenouspreparation of propagating pancreatic cells expressing both CD56 andEphA4, wherein the propagating pancreatic cells have the capacity todifferentiate into insulin producing cells.